Anti-cd38 antibodies and methods of use

ABSTRACT

The disclosure provides binding proteins that bind CD38 polypeptides, e.g., human and cynomolgus monkey CD38 polypeptides. For example, the binding proteins can be monospecific, bispecific, or trispecific binding proteins with at least one antigen binding domain that binds a CD38 polypeptide. The disclosure also provides methods for making binding proteins that bind CD38 polypeptides and uses of such binding proteins.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 62/570,655, filed Oct. 10, 2017; U.S. ProvisionalApplication Ser. No. 62/570,660, filed Oct. 11, 2017; U.S. ProvisionalApplication Ser. No. 62/676,221, filed May 24, 2018; and EP ApplicationNo. EP18187186.4, filed Aug. 3, 2018; all of which are incorporatedherein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 183952029900seqlist.TXT,date recorded: Oct. 9, 2018, size: 158 KB).

FIELD OF THE INVENTION

The disclosure relates to binding proteins that bind CD38 polypeptides(e.g., human and cynomolgus monkey CD38 polypeptides), includingmonospecific, bispecific, or trispecific binding proteins with at leastone antigen binding domain that binds a CD38 polypeptide, as well aspolynucleotides, host cells, methods of production, and methods of userelated thereto.

BACKGROUND

Monoclonal antibody based biotherapeutics have become an importantavenue for new drug development. Monoclonal antibody technology offersspecific targeting, precise signaling delivery and/or payload tospecific cell population, and provides long lasting biological effectthrough its Fc functions. Efforts in antibody engineering have alloweddeveloping multispecific antibodies combining the specificities ofmultiple monoclonal antibodies for various biological applications,expanding the scope of antibody drug development.

CD38 is an attractive drug target, since it is expressed on the cellsurface of a variety of lymphoid tumor cells (see Stevenson, G. T.(2006) Mol. Med. 12:345-346). DARZALEX● (daratumumab) is an anti-CD38antibody approved for use in treating multiple myeloma. However, a needexists for therapeutics targeting CD38 with a different mode of actionand/or improved properties, including but not limited to high affinitybinding to CD38, cross-reactivity between human and cynomolgus monkeyCD38 polypeptides, binding to lymphoma cells (e.g., multiple myelomalarge B-cell lymphoma cell lines), and the ability to induce apoptosisand/or antibody-dependent cell-mediated cytotoxicity (ADCC) and T cellmediated anti-tumor activities.

BRIEF SUMMARY

Provided herein are binding proteins that bind CD38 polypeptides (e.g.,human and cynomolgus monkey CD38 polypeptides), including monospecific,bispecific, or trispecific binding proteins with at least one antigenbinding site that binds a CD38 polypeptide. Advantageously, thesebinding proteins have the ability to recruit T cells to the proximity ofcancer cells, subsequently to activate T cells and promote the activatedT cells killing of adjacent cancer cells through a Granzyme/Perforinmechanism, providing a different mode of action for anti-tumor activityfrom anti-CD38 antibodies such as DARZALEX● (daratumumab). Moreover, theability to bind both human and cynomolgus monkey CD38 polypeptidesallows binding proteins to be readily tested in preclinicaltoxicological studies, e.g., to evaluate their safety profiles for laterclinical use.

In some embodiments, provided herein are monospecific binding proteinsthat bind to a human CD38 polypeptide. In some embodiments, the bindingproteins cross-react with human and cynomolgus monkey CD38 polypeptides.In some embodiments, the binding proteins bind to human isoform A andisoform E CD38 polypeptides. In some embodiments, the binding proteinspossess one or more of the following features (in any combination):binds to the extracellular domain of a human CD38 polypeptide (e.g.,comprising the amino acid sequence of SEQ ID NO:1) as a purifiedprotein, as assayed by SPR; binds to the extracellular domain of a humanCD38 polypeptide (e.g., comprising the amino acid sequence of SEQ IDNO:1) as a purified protein with a K_(D) of 1.5 nM or less, as assayedby SPR; binds to the extracellular domain of a human CD38 polypeptide(e.g., comprising the amino acid sequence of SEQ ID NO:1) expressed onthe surface of a cell, as assayed by flow cytometry; binds to theextracellular domain of a human CD38 polypeptide (e.g., comprising theamino acid sequence of SEQ ID NO:1) expressed on the surface of a cellwith an apparent K_(D) of 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or less, asassayed by flow cytometry; binds to the extracellular domain of acynomolgus monkey CD38 polypeptide (e.g., comprising the amino acidsequence of SEQ ID NO:30) as a purified protein, as assayed by SPR;binds to the extracellular domain of a cynomolgus monkey CD38polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30)as a purified protein with a K_(D) of 3.5 nM or less, as assayed by SPR;binds to the extracellular domain of a cynomolgus monkey CD38polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30)expressed on the surface of a cell, as assayed by flow cytometry; bindsto the extracellular domain of a cynomolgus monkey CD38 polypeptide(e.g., comprising the amino acid sequence of SEQ ID NO:30) expressed onthe surface of a cell with an apparent K_(D) of 7.5 nM or less, asassayed by flow cytometry; binds to the extracellular domain of a humanisoform E CD38 polypeptide (e.g., comprising the amino acid sequence ofSEQ ID NO:105) as a purified protein, as assayed by ELISA; binds to theextracellular domain of a human isoform E CD38 polypeptide (e.g.,comprising the amino acid sequence of SEQ ID NO:105) expressed on thesurface of a cell, as assayed by flow cytometry; induces apoptosis orantibody-dependent cellular cytotoxicity (ADCC) of a cell expressingCD38 on its cell surface; and has one or more mutations (e.g., in an Fcregion) resulting in decreased binding to FcγRI and/or FcγRII, ascompared to the same binding protein without the one or more mutations.For exemplary assays, see Examples 1, 3, and 4. In some embodiments, theKD is measured at 4° C. or 25° C.

In some embodiments, provided herein are trispecific binding proteinsthat bind to a human CD38 polypeptide. In some embodiments, thetrispecific binding proteins bind (e.g., simultaneously) to a CD38polypeptide (e.g., expressed on the surface of a cell) and one or moreother target antigens expressed on the surface of a second cell, therebyrecruiting the second cell in proximity with the cell expressing theCD38 polypeptide. In some embodiments, the trispecific binding proteinsbind (e.g., simultaneously) to a CD38 polypeptide (e.g., expressed onthe surface of a cell) and one or two target antigens expressed on thesurface of a T cell, thereby recruiting the T cell in proximity with thecell expressing the CD38 polypeptide. In some embodiments, thetrispecific binding proteins activate the T cell and/or provide aCD28-mediated costimulatory signal to the T cell. In some embodiments,the trispecific binding proteins cross-react with human and cynomolgusmonkey CD38 polypeptides. In some embodiments, the trispecific bindingproteins bind to human isoform A and isoform E CD38 polypeptides. Insome embodiments, the trispecific binding proteins possess one or moreof the following features (in any combination): binds to theextracellular domain of a human CD38 polypeptide (e.g., comprising theamino acid sequence of SEQ ID NO:1) as a purified protein, as assayed bySPR; binds to the extracellular domain of a human CD38 polypeptide(e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purifiedprotein with a K_(D) of 1.5 nM or less, as assayed by SPR; binds to theextracellular domain of a human CD38 polypeptide (e.g., comprising theamino acid sequence of SEQ ID NO:1) expressed on the surface of a cell,as assayed by flow cytometry; binds to the extracellular domain of ahuman CD38 polypeptide (e.g., comprising the amino acid sequence of SEQID NO:1) expressed on the surface of a cell with an apparent K_(D) of 20nM, 15 nM, 10 nM, 5 nM, 1 nM, or less, as assayed by flow cytometry;binds to the extracellular domain of a cynomolgus monkey CD38polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30)as a purified protein, as assayed by SPR; binds to the extracellulardomain of a cynomolgus monkey CD38 polypeptide (e.g., comprising theamino acid sequence of SEQ ID NO:30) as a purified protein with a K_(D)of 3.5 nM or less, as assayed by SPR; binds to the extracellular domainof a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acidsequence of SEQ ID NO:30) expressed on the surface of a cell, as assayedby flow cytometry; binds to the extracellular domain of a cynomolgusmonkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQID NO:30) expressed on the surface of a cell with an apparent K_(D) of7.5 nM or less, as assayed by flow cytometry; binds to the extracellulardomain of a human isoform E CD38 polypeptide (e.g., comprising the aminoacid sequence of SEQ ID NO:105) as a purified protein, as assayed byELISA; binds to the extracellular domain of a human isoform E CD38polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:105)expressed on the surface of a cell, as assayed by flow cytometry;induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) ofa cell expressing CD38 on its cell surface; and has one or moremutations (e.g., in an Fc region) resulting in decreased binding toFcγRI and/or FcγRII, as compared to the same binding protein without theone or more mutations; induces T cell (e.g., CD4+ and/or CD8+ T cell)proliferation; induces T cell (e.g., CD4+ and/or CD8+ T cell) expressionof Bcl-xL; induces apoptosis of CD38+ cells; binds to CD38 expressed onthe surface of a cell and one or more T cell target antigen(s) expressedon the surface of a T cell; binds to CD38 expressed on the surface of acell, CD28 expressed on the surface of a T cell, and CD3 expressed onthe surface of a T cell; stimulates activation of the T cell receptor;induces costimulation of T cell receptor signaling (e.g., as mediated byCD28); and has one or more mutations (e.g., in an Fc region) resultingin decreased induction of cytokine release (e.g., IFN-γ, IL-2, and/orTNF-α) by PBMCs, as compared to the same binding protein without the oneor more mutations; induces cytokine release (e.g., IFN-γ and/or IL-6) byPBMCs in the presence of a CD38+ target cell. For exemplary assays, seeExamples 1, 3, and 4. In some embodiments, the KD is measured at 4° C.or 25° C.

In some embodiments, provided herein is a binding protein comprising anantigen binding site that binds a CD38 poly peptide, wherein the antigenbinding site comprises: (a) an antibody heavy chain variable (VH) domaincomprising a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) orIYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acidsequence of ARTGGLRRAYFTY (SEQ ID NO:33); and/or (b) an antibody lightchain variable (VL) domain comprising a CDR-L1 sequence comprising theamino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ IDNO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments,the antigen binding site comprises: (a) an antibody heavy chain variable(VH) domain comprising a CDR-H1 sequence comprising the amino acidsequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32)or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the aminoacid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and/or (b) an antibodylight chain variable (VL) domain comprising a CDR-L1 sequence comprisingthe amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS(SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprisingthe amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In someembodiments, the antigen binding site comprises: (a) an antibody heavychain variable (VH) domain comprising a CDR-H1 sequence comprising theamino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and aCDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQID NO:33); and/or (b) an antibody light chain variable (VL) domaincomprising a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acidsequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments,the VH domain comprises the sequence, from N-terminus to C-terminus,FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; wherein FR1 comprises the sequenceQVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQID NO:87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); wherein FR2comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) orMHWVKEAPGQGLEWIGY (SEQ ID NO:91); wherein FR3 comprises the sequenceNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) orNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and wherein FR4comprises the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments,the VH domain comprises the amino acid sequence of SEQ ID NO:5, and/orthe VL domain comprises the amino acid sequence of SEQ ID NO:6. In someembodiments, the binding protein comprises an antibody heavy chaincomprising the amino acid sequence of SEQ ID NO:7 and an antibody lightchain comprising the amino acid sequence of SEQ ID NO:8. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO:17, and/or the VL domain comprises the amino acid sequence of SEQ IDNO:18. In some embodiments, the binding protein comprises an antibodyheavy chain comprising the amino acid sequence of SEQ ID NO:19 and anantibody light chain comprising the amino acid sequence of SEQ ID NO:20.In some embodiments, the VH domain comprises the amino acid sequence ofSEQ ID NO:21, and/or the VL domain comprises the amino acid sequence ofSEQ ID NO:18. In some embodiments, the binding protein comprises anantibody heavy chain comprising the amino acid sequence of SEQ ID NO:22and an antibody light chain comprising the amino acid sequence of SEQ IDNO:20. In some embodiments, the VH domain comprises the amino acidsequence of SEQ ID NO:23, and/or the VL domain comprises the amino acidsequence of SEQ ID NO:18. In some embodiments, the binding proteincomprises an antibody heavy chain comprising the amino acid sequence ofSEQ ID NO:24 and an antibody light chain comprising the amino acidsequence of SEQ ID NO:20. In some embodiments, the antigen binding sitecomprises: (a) an antibody heavy chain variable (VH) domain comprising aCDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ IDNO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT(SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequenceof ARTGGLRRAYFTY (SEQ ID NO:33); and (b) an antibody light chainvariable (VL) domain comprising a CDR-L1 sequence comprising the aminoacid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprisingthe amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In someembodiments, the VH domain comprises the sequence, from N-terminus toC-terminus, FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; wherein FR1 comprisesthe sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86),QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87), or QVQLVQSGAEVVKPGASVKMSCKAS(SEQ ID NO:88); wherein FR2 comprises the sequence MHWVKEAPGQRLEWIGY(SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO:91); wherein FR3comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ IDNO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); andwherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:96). In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO:13, and/or the VL domain comprises the amino acid sequence of SEQ IDNO:14. In some embodiments, the binding protein comprises an antibodyheavy chain comprising the amino acid sequence of SEQ ID NO:15 and anantibody light chain comprising the amino acid sequence of SEQ ID NO:16.

In some embodiments, provided herein is a binding protein comprising anantigen binding site that binds a CD38 polypeptide, wherein the antigenbinding site comprises: (a) an antibody heavy chain variable (VH) domaincomprising a CDR-H1 sequence comprising the amino acid sequence ofGFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acidsequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprisingthe amino acid sequence of ARMFRGAFDY (SEQ ID NO:43); and (b) anantibody light chain variable (VL) domain comprising a CDR-L1 sequencecomprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and aCDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ IDNO:46). In some embodiments, the VH domain comprises the amino acidsequence of SEQ ID NO:9, and/or the VL domain comprises the amino acidsequence of SEQ ID NO:10. In some embodiments, the binding proteincomprises an antibody heavy chain comprising the amino acid sequence ofSEQ ID NO:11 and an antibody light chain comprising the amino acidsequence of SEQ ID NO:12. In some embodiments, the antigen binding sitecross-reacts with an extracellular domain of a human CD38 polypeptideand an extracellular domain of a cynomolgus monkey CD38 polypeptide. Insome embodiments, the antigen binding site binds a human CD38polypeptide comprising the amino acid sequence of SEQ ID NO:1. In someembodiments, the antigen binding site binds the human CD38 polypeptidecomprising the amino acid sequence of SEQ ID NO:1 with an equilibriumdissociation constant (K_(D)) of 2.1 nM or less. In some embodiments,the antigen binding site binds a human isoform E CD38 polypeptidecomprising the amino acid sequence of SEQ ID NO:105. In someembodiments, the antigen binding site binds a cynomolgus monkey CD38polypeptide comprising the amino acid sequence of SEQ ID NO:30. In someembodiments, the antigen binding site binds the cynomolgus monkey CD38polypeptide comprising the amino acid sequence of SEQ ID NO:30 with anequilibrium dissociation constant (K_(D)) of 1.3 nM or less.

In some embodiments of any of the above embodiments, the binding proteinis a chimeric or humanized antibody. In some embodiments, the bindingprotein is a human antibody. In some embodiments, the binding protein isa monoclonal antibody. In some embodiments, the binding proteincomprises one or more full-length antibody heavy chains comprising an Fcregion. In some embodiments, the Fc region is a human Fc regioncomprising one or more mutations that reduce or eliminate Fc receptorbinding and/or effector function of the Fc region. In some embodiments,the Fc region is a human IgG1 Fc region. In some embodiments, the humanIgG1 Fc region comprises amino acid substitutions at positionscorresponding to positions 234, 235, and 329 of human IgG1 according toEU Index, wherein the amino acid substitutions are L234A, L235A, andP329A. In some embodiments, the human IgG1 Fc region comprises aminoacid substitutions at positions corresponding to positions 298, 299, and300 of human IgG1 according to EU Index, wherein the amino acidsubstitutions are S298N, T299A, and Y300S. In some embodiments, the Fcregion is a human IgG4 Fc region. In some embodiments, the human IgG4 Fcregion comprises amino acid substitutions at positions corresponding topositions 228 and 409 of human IgG4 according to EU Index, wherein theamino acid substitutions are S228P and R409K. In some embodiments, thehuman IgG4 Fc region comprises amino acid substitutions at positionscorresponding to positions 234 and 235 of human IgG4 according to EUIndex, wherein the amino acid substitutions are F234A and L235A. In someembodiments, the human IgG4 Fc region comprises amino acid substitutionsat positions corresponding to positions 233-236 of human IgG4 accordingto EU Index, wherein the amino acid substitutions are E233P, F234V,L235A, and a deletion at 236. In some embodiments, the human IgG4 Fcregion comprises amino acid substitutions at positions corresponding topositions 233-237 of human IgG4 according to EU Index, wherein thesequence EFLGG is replaced by PVAG. In some embodiments, the bindingprotein comprises an antibody F(ab), F(ab′)2, Fab′-SH, Fv, or scFvfragment. In some embodiments, the binding protein is conjugated to acytotoxic agent or label. In some embodiments, the binding protein is abispecific binding protein comprising the first antigen binding sitethat binds the CD38 polypeptide and a second antigen binding site. Insome embodiments, the binding protein is a trispecific binding proteincomprising the first antigen binding site that binds the CD38polypeptide, a second antigen binding site, and a third antigen bindingsite. In some embodiments, the first antigen binding site binds theextracellular domain of a human CD38 polypeptide, and wherein the secondand third antigen binding sites each bind a T-cell surface protein. Insome embodiments, the first antigen binding site binds the extracellulardomain of a human CD38 polypeptide, and wherein (a) the second antigenbinding site binds a human CD28 polypeptide, and the third antigenbinding site binds a human CD3 polypeptide, or (b) the second antigenbinding site binds a human CD3 polypeptide, and the third antigenbinding site binds a human CD28 polypeptide.

In some embodiments, provided herein is a binding protein comprisingthree antigen binding sites that each bind one or more target proteins,wherein at least one of the three antigen binding sites cross-reactswith an extracellular domain of a human CD38 polypeptide and anextracellular domain of a cynomolgus monkey CD38 polypeptide. In someembodiments, the binding protein cross-reacts with a human CD38polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:105. In some embodiments, the binding protein cross-reacts with acynomolgus monkey CD38 polypeptide comprising the amino acid sequence ofSEQ ID NO:30. In some embodiments, the binding protein comprises anantigen binding site that cross-reacts with an extracellular domain of ahuman CD38 polypeptide and an extracellular domain of a cynomolgusmonkey CD38 polypeptide and two antigen binding sites that each bind aT-cell surface protein. In some embodiments, the binding proteincomprises an antigen binding site that cross-reacts with anextracellular domain of a human CD38 polypeptide and an extracellulardomain of a cynomolgus monkey CD38 polypeptide, an antigen binding sitethat binds a human CD28 polypeptide, and an antigen binding site thatbinds a human CD3 polypeptide. In some embodiments, the binding proteincomprises four polypeptide chains that form the three antigen bindingsites, wherein a first polypeptide chain comprises a structurerepresented by the formula:

V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I]

and a second polypeptide chain comprises a structure represented by theformula:

V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3)  [II]

and a third polypeptide chain comprises a structure represented by theformula:

V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3)  [III]

and a fourth polypeptide chain comprises a structure represented by theformula:

V_(L3)-C_(L)  [IV]

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(L3) is a third immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

V_(H3) is a third immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H1) is an immunoglobulin C_(H1) heavy chain constant domain;

C_(H2) is an immunoglobulin C_(H2) heavy chain constant domain;

C_(H3) is an immunoglobulin C_(H3) heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the C_(H1) and C_(H2)domains; and

L₁, L₂, L₃ and L₄ are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula IIform a cross-over light chain-heavy chain pair, and

wherein: (a) the V_(H1) domain comprises a CDR-H1 sequence comprisingthe amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ IDNO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT(SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequencecomprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), andthe V_(L1) domain comprises a CDR-L1 sequence comprising the amino acidsequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), aCDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35)or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acidsequence of QQNKEDPWT (SEQ ID NO:36);

(b) the V_(H2) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), aCDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ IDNO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising theamino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L2)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36); or(c) the V_(H3) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), aCDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ IDNO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising theamino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding proteincomprises four polypeptide chains that form the three antigen bindingsites, wherein a first polypeptide chain comprises a structurerepresented by the formula:

V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I]

and a second polypeptide chain comprises a structure represented by theformula:

V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3)  [II]

and a third polypeptide chain comprises a structure represented by theformula:

V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3)  [III]

and a fourth polypeptide chain comprises a structure represented by theformula:

V_(L3)-C_(L)  [IV]

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(L3) is a third immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

V_(H3) is a third immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H1) is an immunoglobulin C_(H1) heavy chain constant domain;

C_(H2) is an immunoglobulin C_(H2) heavy chain constant domain;

C_(H3) is an immunoglobulin C_(H3) heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the C_(H1) and C_(H2)domains; and

L₁, L₂, L₃ and L₄ are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula IIform a cross-over light chain-heavy chain pair, and

wherein: (a) the V_(H1) domain comprises a CDR-H1 sequence comprisingthe amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ IDNO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT(SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequencecomprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), andthe V_(L1) domain comprises a CDR-L1 sequence comprising the amino acidsequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), aCDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35)or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acidsequence of QQNKEDPWT (SEQ ID NO:36);

(b) the V_(H2) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), aCDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ IDNO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising theamino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L2)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36); or(c) the V_(H3) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), aCDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ IDNO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising theamino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H1) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acidsequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L1)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acidsequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36); the V_(H1) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L1)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofQSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acidsequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36); the V_(H2) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acidsequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L2)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acidsequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36); the V_(H2) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L2)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofQSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acidsequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36); the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acidsequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acidsequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36); or the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofQSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acidsequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments,the V_(H3) domain comprises a CDR-H1 sequence comprising the amino acidsequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising theamino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequencecomprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), andthe V_(L3) domain comprises a CDR-L1 sequence comprising the amino acidsequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising theamino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or theV_(H3) domain comprises a CDR-H1 sequence comprising the amino acidsequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising theamino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequencecomprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), andthe V_(L3) domain comprises a CDR-L1 sequence comprising the amino acidsequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising theamino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In someembodiments, the V_(H3) domain comprises the amino acid sequence of SEQID NO:5, and the V_(L3) domain comprises the amino acid sequence of SEQID NO:6; the V_(H3) domain comprises the amino acid sequence of SEQ IDNO:17, and the V_(L3) domain comprises the amino acid sequence of SEQ IDNO:18; the V_(H3) domain comprises the amino acid sequence of SEQ IDNO:21, and the V_(L3) domain comprises the amino acid sequence of SEQ IDNO:18; the V_(H3) domain comprises the amino acid sequence of SEQ IDNO:23, and the V_(L3) domain comprises the amino acid sequence of SEQ IDNO:18; or the V_(H3) domain comprises the amino acid sequence of SEQ IDNO:13, and the V_(L3) domain comprises the amino acid sequence of SEQ IDNO:14. In some embodiments, the binding protein comprises fourpolypeptide chains that form the three antigen binding sites, wherein afirst polypeptide chain comprises a structure represented by theformula:

V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I]

and a second polypeptide chain comprises a structure represented by theformula:

V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3)  [II]

and a third polypeptide chain comprises a structure represented by theformula:

V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3)  [III]

and a fourth polypeptide chain comprises a structure represented by theformula:

V_(L3)-C_(L)  [IV]

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(L3) is a third immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

V_(H3) is a third immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H1) is an immunoglobulin C_(H1) heavy chain constant domain;

C_(H2) is an immunoglobulin C_(H2) heavy chain constant domain;

C_(H3) is an immunoglobulin C_(H3) heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the C_(H1) and C_(H2)domains; and

L₁, L₂, L₃ and L₄ are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula IIform a cross-over light chain-heavy chain pair, and

wherein (a) the V_(H1) domain comprises a CDR-H1 sequence comprising theamino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequencecomprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and aCDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ IDNO:43), and the V_(L1) domain comprises a CDR-L1 sequence comprising theamino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequencecomprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46);

(b) the V_(H2) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprisingthe amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ IDNO:43), and the V_(L2) domain comprises a CDR-L1 sequence comprising theamino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequencecomprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46);or(c) the V_(H3) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprisingthe amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ IDNO:43), and the V_(L3) domain comprises a CDR-L1 sequence comprising theamino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequencecomprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46).In some embodiments, the V_(H3) domain comprises a CDR-H1 sequencecomprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42),and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY(SEQ ID NO:43), and the V_(L3) domain comprises a CDR-L1 sequencecomprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and aCDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ IDNO:46). In some embodiments, the V_(H3) domain comprises the amino acidsequence of SEQ ID NO:9, and the V_(L3) domain comprises the amino acidsequence of SEQ ID NO:10. In some embodiments, the V_(H1) domaincomprises the amino acid sequence of SEQ ID NO:49, the V_(L1) domaincomprises the amino acid sequence of SEQ ID NO:50, the V_(H2) domaincomprises the amino acid sequence of SEQ ID NO:53, and the V_(L2) domaincomprises the amino acid sequence of SEQ ID NO:54; the V_(H2) domaincomprises the amino acid sequence of SEQ ID NO:49, the V_(L2) domaincomprises the amino acid sequence of SEQ ID NO:50, the V_(H1) domaincomprises the amino acid sequence of SEQ ID NO:53, and the V_(L1) domaincomprises the amino acid sequence of SEQ ID NO:54; the V_(H1) domaincomprises the amino acid sequence of SEQ ID NO:51, the V_(L1) domaincomprises the amino acid sequence of SEQ ID NO:52, the V_(H2) domaincomprises the amino acid sequence of SEQ ID NO:53, and the V_(L2) domaincomprises the amino acid sequence of SEQ ID NO:54; the V_(H2) domaincomprises the amino acid sequence of SEQ ID NO:51, the V_(L2) domaincomprises the amino acid sequence of SEQ ID NO:52, the V_(H1) domaincomprises the amino acid sequence of SEQ ID NO:53, and the V_(L1) domaincomprises the amino acid sequence of SEQ ID NO:54 the V_(H1) domaincomprises the amino acid sequence of SEQ ID NO:49, the V_(L1) domaincomprises the amino acid sequence of SEQ ID NO:50, the V_(H2) domaincomprises the amino acid sequence of SEQ ID NO:84, and the V_(L2) domaincomprises the amino acid sequence of SEQ ID NO:85; the V_(H2) domaincomprises the amino acid sequence of SEQ ID NO:49, the V_(L2) domaincomprises the amino acid sequence of SEQ ID NO:50, the V_(H1) domaincomprises the amino acid sequence of SEQ ID NO:84, and the V_(L1) domaincomprises the amino acid sequence of SEQ ID NO:85; the V_(H1) domaincomprises the amino acid sequence of SEQ ID NO:51, the V_(L1) domaincomprises the amino acid sequence of SEQ ID NO:52, the V_(H2) domaincomprises the amino acid sequence of SEQ ID NO:84, and the V_(L2) domaincomprises the amino acid sequence of SEQ ID NO:85; or the V_(H2) domaincomprises the amino acid sequence of SEQ ID NO:51, the V_(L2) domaincomprises the amino acid sequence of SEQ ID NO:52, the V_(H1) domaincomprises the amino acid sequence of SEQ ID NO:84, and the V_(L1) domaincomprises the amino acid sequence of SEQ ID NO:85. In some embodiments,the V_(H1) domain comprises the amino acid sequence of SEQ ID NO:49, theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:50, theV_(H2) domain comprises the amino acid sequence of SEQ ID NO:53, theV_(L2) domain comprises the amino acid sequence of SEQ ID NO:54, theV_(H3) domain comprises the amino acid sequence of SEQ ID NO:13, and theV_(L3) domain comprises the amino acid sequence of SEQ ID NO:14; or theV_(H1) domain comprises the amino acid sequence of SEQ ID NO:49, theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:50, theV_(H2) domain comprises the amino acid sequence of SEQ ID NO:53, theV_(L2) domain comprises the amino acid sequence of SEQ ID NO:54, theV_(H3) domain comprises the amino acid sequence of SEQ ID NO:9, and theV_(L3) domain comprises the amino acid sequence of SEQ ID NO:10. In someembodiments, at least one of L₁, L₂, L₃ or L₄ is independently 0 aminoacids in length. In some embodiments, L₁, L₂, L₃ or L₄ are eachindependently at least one amino acid in length. In some embodiments,(a) L₁, L₂, L₃ and L₄ each independently are zero amino acids in lengthor comprise a sequence selected from the group consisting of GGGGSGGGGS(SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ IDNO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59); or (b)L₁, L₂, L₃ and L₄ each independently comprise a sequence selected fromthe group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), andGGSGSSGSGG (SEQ ID NO:59). In some embodiments, L₁ comprises thesequence GQPKAAP (SEQ ID NO:58), L₂ comprises the sequence TKGPS (SEQ IDNO:57), L₃ comprises the sequence S, and L₄ comprises the sequence RT;L₁ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₂ comprises thesequence GGGGSGGGGS (SEQ ID NO:55), L₃ is 0 amino acids in length, andL₄ is 0 amino acids in length; L₁ comprises the sequence GGSGSSGSGG (SEQID NO:59), L₂ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₃ is 0amino acids in length, and L₄ is 0 amino acids in length; or L₁comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L₂ is 0 aminoacids in length, L₃ comprises the sequence GGGGSGGGGSGGGGS (SEQ IDNO:56), and L₄ is 0 amino acids in length. In some embodiments, thehinge-C_(H2)-C_(H3) domains of the second and the third polypeptidechains are human IgG4 hinge-C_(H2)-C_(H3) domains, and wherein thehinge-C_(H2)-C_(H3) domains each comprise amino acid substitutions atpositions corresponding to positions 234 and 235 of human IgG4 accordingto EU Index, wherein the amino acid substitutions are F234A and L235A.In some embodiments, the hinge-C_(H2)-C_(H3) domains of the second andthe third polypeptide chains are human IgG4 hinge-C_(H2)-C_(H3) domains,and wherein the hinge-C_(H2)-C_(H3) domains each comprise amino acidsubstitutions at positions corresponding to positions 233-236 of humanIgG4 according to EU Index, wherein the amino acid substitutions areE233P, F234V, L235A, and a deletion at 236. In some embodiments, thehinge-C_(H2)-C_(H3) domains of the second and the third polypeptidechains are human IgG4 hinge-C_(H2)-C_(H3) domains, and wherein thehinge-C_(H2)-C_(H3) domains each comprise amino acid substitutions atpositions corresponding to positions 228 and 409 of human IgG4 accordingto EU Index, wherein the amino acid substitutions are S228P and R409K.In some embodiments, the hinge-C_(H2)-C_(H3) domains of the second andthe third polypeptide chains are human IgG1 hinge-C_(H2)-C_(H3) domains,and wherein the hinge-C_(H2)-C_(H3) domains each comprise amino acidsubstitutions at positions corresponding to positions 234, 235, and 329of human IgG1 according to EU Index, wherein the amino acidsubstitutions are L234A, L235A, and P329A. In some embodiments, thehinge-C_(H2)-C_(H3) domains of the second and the third polypeptidechains are human IgG1 hinge-C_(H2)-C_(H3) domains, and wherein thehinge-C_(H2)-C_(H3) domains each comprise amino acid substitutions atpositions corresponding to positions 298, 299, and 300 of human IgG1according to EU Index, wherein the amino acid substitutions are S298N,T299A, and Y300S. In some embodiments, the hinge-C_(H2)-C_(H3) domain ofthe second polypeptide chain comprises amino acid substitutions atpositions corresponding to positions 349, 366, 368, and 407 of humanIgG1 or IgG4 according to EU Index, wherein the amino acid substitutionsare Y349C, T366S, L368A, and Y407V; and wherein the hinge-C_(H2)-C_(H3)domain of the third polypeptide chain comprises amino acid substitutionsat positions corresponding to positions 354 and 366 of human IgG1 orIgG4 according to EU Index, wherein the amino acid substitutions areS354C and T366W. In some embodiments, the hinge-C_(H2)-C_(H3) domain ofthe second polypeptide chain comprises amino acid substitutions atpositions corresponding to positions 354 and 366 of human IgG1 or IgG4according to EU Index, wherein the amino acid substitutions are S354Cand T366W; and wherein the hinge-C_(H2)-C_(H3) domain of the thirdpolypeptide chain comprises amino acid substitutions at positionscorresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4according to EU Index, wherein the amino acid substitutions are Y349C,T366S, L368A, and Y407V. In some embodiments, the first polypeptidechain comprises the amino acid sequence of SEQ ID NO:61, the secondpolypeptide chain comprises the amino acid sequence of SEQ ID NO:60, thethird polypeptide chain comprises the amino acid sequence of SEQ IDNO:62, and the fourth polypeptide chain comprises the amino acidsequence of SEQ ID NO:63. In some embodiments, the first polypeptidechain comprises the amino acid sequence of SEQ ID NO:61, the secondpolypeptide chain comprises the amino acid sequence of SEQ ID NO:64, thethird polypeptide chain comprises the amino acid sequence of SEQ IDNO:65, and the fourth polypeptide chain comprises the amino acidsequence of SEQ ID NO:63. In some embodiments, the first polypeptidechain comprises the amino acid sequence of SEQ ID NO:61, the secondpolypeptide chain comprises the amino acid sequence of SEQ ID NO:66, thethird polypeptide chain comprises the amino acid sequence of SEQ IDNO:67, and the fourth polypeptide chain comprises the amino acidsequence of SEQ ID NO:63. In some embodiments, the first polypeptidechain comprises the amino acid sequence of SEQ ID NO:61, the secondpolypeptide chain comprises the amino acid sequence of SEQ ID NO:60, thethird polypeptide chain comprises the amino acid sequence of SEQ IDNO:68, and the fourth polypeptide chain comprises the amino acidsequence of SEQ ID NO:69. In some embodiments, the first polypeptidechain comprises the amino acid sequence of SEQ ID NO:61, the secondpolypeptide chain comprises the amino acid sequence of SEQ ID NO:64, thethird polypeptide chain comprises the amino acid sequence of SEQ IDNO:70, and the fourth polypeptide chain comprises the amino acidsequence of SEQ ID NO:69. In some embodiments, the first polypeptidechain comprises the amino acid sequence of SEQ ID NO:61, the secondpolypeptide chain comprises the amino acid sequence of SEQ ID NO:66, thethird polypeptide chain comprises the amino acid sequence of SEQ IDNO:71, and the fourth polypeptide chain comprises the amino acidsequence of SEQ ID NO:69.

In some embodiments, provided herein is a binding protein comprisingthree antigen binding sites that each bind one or more target proteins,wherein the binding protein comprises four polypeptide chains that formthe three antigen binding sites, wherein a first polypeptide chaincomprises a structure represented by the formula:

V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I]

and a second polypeptide chain comprises a structure represented by theformula:

V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3)  [II]

and a third polypeptide chain comprises a structure represented by theformula:

V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3)  [III]

and a fourth polypeptide chain comprises a structure represented by theformula:

V_(L3)-C_(L)  [IV]

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(L3) is a third immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

V_(H3) is a third immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H1) is an immunoglobulin C_(H1) heavy chain constant domain;

C_(H2) is an immunoglobulin C_(H2) heavy chain constant domain;

C_(H3) is an immunoglobulin C_(H3) heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the C_(H1) and C_(H2)domains; and

L₁, L₂, L₃ and L₄ are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula IIform a cross-over light chain-heavy chain pair; and

wherein (a) the hinge-C_(H2)-C_(H3) domains of the second and the thirdpolypeptide chains are human IgG1 hinge-C_(H2)-C_(H3) domains, andwherein the hinge-C_(H2)-C_(H3) domains each comprise amino acidsubstitutions at positions corresponding to positions 298, 299, and 300of human IgG1 according to EU Index, wherein the amino acidsubstitutions are S298N, T299A, and Y300S; or (b) thehinge-C_(H2)-C_(H3) domains of the second and the third polypeptidechains are human IgG4 hinge-C_(H2)-C_(H3) domains, and wherein thehinge-C_(H2)-C_(H3) domains each comprise amino acid substitutions atpositions corresponding to positions 233-236 of human IgG4 according toEU Index, wherein the amino acid substitutions are E233P, F234V, L235A,and a deletion at 236. In some embodiments, the human IgG4hinge-C_(H2)-C_(H3) domains comprise amino acid substitutions atpositions corresponding to positions 233-237 of human IgG4 according toEU Index, wherein the sequence EFLGG is replaced by PVAG. In someembodiments, at least one pair of V_(H1) and V_(L1), V_(H2) and V_(L2),and V_(H3) and V_(L3) forms an antigen binding site that binds a CD38polypeptide. In some embodiments, one, two, or now abandoned three pairsof V_(H1) and V_(L1), V_(H2) and V_(L2), and V_(H3) and V_(L3) form anantigen binding site that binds an antigen target selected from thegroup consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK,BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2,CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21,CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27,CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152,CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1,chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12,CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1,EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF,Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1,IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8,IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17,IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3,LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40,OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2,STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5,TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF,VISTA, Vstm3, WUCAM, and XCR1. In some embodiments, a first pair ofV_(H1) and V_(L1), V_(H2) and V_(L2), and V_(H3) and V_(L3) forms anantigen binding site that binds a human CD3 polypeptide, a second pairof V_(H1) and V_(L1), V_(H2) and V_(L2), and V_(H3) and V_(L3) forms anantigen binding site that binds a human CD28 polypeptide, and a thirdpair of V_(H1) and V_(L1), V_(H2) and V_(L2), and V_(H3) and V_(L3)forms an antigen binding site that binds a human antigen target selectedfrom the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA,BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B74, C3, C5,CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20,CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24,CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L,CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279,CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1,CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphatediphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24,GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE,IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R,IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1,IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35,ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1,NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152,SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3,TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP,TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.

In some embodiments, provided herein is a kit of polynucleotides,comprising: (a) a first polynucleotide comprising the sequence of SEQ IDNO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72,a third polynucleotide comprising the sequence of SEQ ID NO:74, and afourth polynucleotide comprising the sequence of SEQ ID NO:75; (b) afirst polynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:76, a thirdpolynucleotide comprising the sequence of SEQ ID NO:77, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:75; (c) a firstpolynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:78, a thirdpolynucleotide comprising the sequence of SEQ ID NO:79, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:75; (d) a firstpolynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:72, a thirdpolynucleotide comprising the sequence of SEQ ID NO:80, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:81; (e) a firstpolynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:76, a thirdpolynucleotide comprising the sequence of SEQ ID NO:82, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:81; or (f) a firstpolynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:78, a thirdpolynucleotide comprising the sequence of SEQ ID NO:83, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:81.

In some embodiments, provided herein is a polynucleotide comprising thebinding protein of any one of the above embodiments. In someembodiments, provided herein is a vector comprising a polynucleotidecomprising the binding protein of any one of the above embodiments.

In some embodiments, provided herein is a host cell comprising the kitof polynucleotides, polynucleotide, or vector of any one of the aboveembodiments. In some embodiments, provided herein is a method ofproducing a binding protein, the method comprising culturing the hostcell of any one of the above embodiments such that the binding proteinis produced. In some embodiments, the method further comprisesrecovering the binding protein from the host cell.

In some embodiments, provided herein is a pharmaceutical compositioncomprising the binding protein of any one of the above embodiments and apharmaceutically acceptable carrier.

In some embodiments, provided herein is a method of preventing and/ortreating cancer in a patient comprising administering to the patient atherapeutically effective amount of at least one binding protein of anyone of the above embodiments or the pharmaceutical composition of anyone of the above embodiments. In some embodiments, the binding proteinis a trispecific binding protein comprising a first antigen binding sitethat binds CD3, a second antigen binding site that binds CD28, and athird antigen binding site that binds the extracellular domain of ahuman CD38 polypeptide. In some embodiments, the at least one bindingprotein is co-administered with a chemotherapeutic agent. In someembodiments, the cancer is multiple myeloma. In some embodiments, thecancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia(ALL), chronic lymphocytic leukemia (CLL), or a B cell lymphoma. In someembodiments, the patient is a human. In some embodiments, the patient isselected for treatment because cells of the cancer express a human CD38isoform E polypeptide (e.g., as set forth in SEQ ID NO:105) on theircell surface. In some embodiments, the cancer cells express CD38 andCD28. In some embodiments, the cancer cells express CD38 and do notexpress CD28.

In some embodiments, provided herein is at least one binding protein ofany one of the above embodiments or the pharmaceutical composition ofany one of the above embodiments for use in preventing and/or treatingcancer in a patient (e.g., a patient in need thereof, such as a patientwith cancer). In some embodiments, provided herein is at least onebinding protein of any one of the above embodiments or thepharmaceutical composition of any one of the above embodiments for usein the manufacture of a medicament for preventing and/or treating cancerin a patient (e.g., a patient in need thereof, such as a patient withcancer). In some embodiments of any of the above embodiments, thebinding protein is a trispecific binding protein comprising a firstantigen binding site that binds CD3, a second antigen binding site thatbinds CD28, and a third antigen binding site that binds theextracellular domain of a human CD38 poly peptide. In some embodiments,the at least one binding protein is to be co-administered with achemotherapeutic agent. In some embodiments, the cancer is multiplemyeloma. In some embodiments, the cancer is acute myeloid leukemia(AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia(CLL), or a B cell lymphoma. In some embodiments, the patient is ahuman. In some embodiments, the patient is selected for treatmentbecause cells of the cancer express a human CD38 isoform E polypeptide(e.g., as set forth in SEQ ID NO:105) on their cell surface. In someembodiments, the cancer cells express CD38 and CD28. In someembodiments, the cancer cells express CD38 and do not express CD28.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows the binding of anti-CD38 antibodies mAb1 (top) andisatuximab (bottom) to SU-DHL-8 human lymphoma cells or MOLP-8 humanmultiple myeloma cells using flow cytometry.

FIG. 1B shows the results of flow cytometry binding assays examiningbinding of anti-CD38 antibodies mAb1 or isatuximab (no binding observed)to cells expressing cynomolgus monkey CD38 on their surface.

FIGS. 2A-2I show the results of assays characterizing the binding ofanti-CD38 antibodies to human and cynomolgus monkey CD38 polypeptides.FIG. 2A shows the binding of humanized anti-CD38 antibody mAb2 tosoluble human CD38 (top, “hCD38::Histag”) or cynomolgus monkey CD38(top, “cynoCD38::Histag”) by ELISA, as well as the binding of mAb2 tothe surface of cells expressing human CD83 (bottom, as indicated) orcynomolgus monkey CD38 (bottom, as indicated) by flow cytometry. FIG. 2Bshows the binding of mAb2 to human CD38 (top) or cynomolgus monkey CD38(bottom) by surface plasmon resonance (SPR). FIG. 2C shows the bindingof humanized anti-CD38 antibody mAb3 to soluble human CD38 (top,“hCD38::Histag”) or cynomolgus monkey CD38 (top, “cynoCD38::Histag”) byELISA, as well as the binding of mAb3 to the surface of cells expressinghuman CD83 (bottom, as indicated) or cynomolgus monkey CD38 (bottom, asindicated) by flow cytometry. FIG. 2D shows the binding of mAb3 to humanCD38 (top) or cynomolgus monkey CD38 (bottom) by SPR. FIG. 2E shows thebinding of humanized anti-CD38 antibody mAb5 to soluble human CD38 (top,“hCD38::Histag”) or cynomolgus monkey CD38 (top, “cynoCD38::Histag”) byELISA, as well as the binding of mAb5 to the surface of cells expressinghuman CD83 (bottom, as indicated) or cynomolgus monkey CD38 (bottom, asindicated) by flow cytometry. FIG. 2F shows the binding of mAb5 to humanCD38 (top) or cynomolgus monkey CD38 (bottom) by SPR. FIG. 2G shows thebinding of human anti-CD38 antibody hhy1370 to soluble human CD38 (top,“hCD38::Histag”) or cynomolgus monkey CD38 (top, “cynoCD38::Histag”) byELISA, as well as the binding of hhy1370 to the surface of cellsexpressing human CD83 (bottom, as indicated) or cynomolgus monkey CD38(bottom, as indicated) by flow cytometry. FIG. 2H shows the binding ofhhy1370 to human CD38 (top) or cynomolgus monkey CD38 (bottom) by SPR.FIG. 2I summarizes the binding data from the ELISA, SPR, and FACSexperiments, as well as the percentage identity of each antibody VH(“H”) or VL (“L”) domain to human V region sequence from top to bottomit corresponds to mAb2, mAb3, mAb 4, mAb 5, and mAb6 last line (1195HHKK-3 has not been described by its VL/VH amino acid sequence in thetext below).

FIG. 2J shows the concentration-dependent induction of SU-DHL-8 cellsapoptosis by mAb7 and mAb1 after incubation for 72 hours at 37° C.

FIG. 2K shows antibody-dependent cell-mediated cytotoxicity (ADCC)activity of isatuximab and mAb1 against SU-DHL-8 cells in the presenceof NK92 cells.

FIG. 2L shows concentration-dependent antibody-dependent cell-mediatedcytotoxicity (ADCC) activity of isatuximab (right) and mAb (left)against SU-DHL-8 cells in the presence of NK92 cells after 4 hours at37° C.

FIGS. 2M-2Q showed the results of apoptosis induction assays using theindicated anti-CD38 antibodies against SU-DHL-8 tumor cells. Apoptosiswas quantified by measuring dual Annexin V and Propidium iodide uptakevia flow cytometry. FIG. 2M shows the percentage of apoptotic cellsinduced by each antibody. FIGS. 2N-2Q show dose-dependent induction ofapoptosis in in SU-DHL-8 lymphoma cells by anti-CD38 antibodies mAb2(FIG. 2N), mAb3 (FIG. 2O), mAb4 (FIG. 2P), and mAb5 (FIG. 2Q), as wellas the IC50 for each antibody.

FIG. 3A provides a schematic representation of a trispecific bindingprotein comprising four polypeptide chains that form three antigenbinding sites that binds three target proteins: CD28, CD3, and CD38. Afirst pair of polypeptides possess dual variable domains having across-over orientation (VH1-VH2 and VL2-VL1) forming two antigen bindingsites that recognize CD3 and CD28, and a second pair of polypeptidespossess a single variable domain (VH3 and VL3) forming a single antigenbinding site that recognizes CD38. The trispecific binding protein shownin FIG. 3A uses an IgG4 constant region with a “knobs-into-holes”mutation, where the knob is on the second pair of polypeptides with asingle variable domain.

FIG. 3B provides a schematic representation of an SPR-based assay forexamining the ability of each antigen binding domain ofanti-CD38/anti-CD28/anti-CD3 trispecific binding proteins to bind itscognate antigen.

FIG. 3C shows the results of SPR-based assays for examining CD38 bindingto anti-CD38/anti-CD28/anti-CD3 trispecific binding proteins. Binding ofhuman CD38 to trispecific binding proteins was examined alone (topleft), after pre-binding with CD3 (top center), after pre-binding withCD28 (top right), after pre-binding to CD3 then CD28 (bottom left), orafter pre-binding to CD28 then CD3 (bottom right).

FIG. 4 shows the sequential binding of human CD3, CD28, and CD38polypeptides to anti-CD38/anti-CD28/anti-CD3 trispecific bindingproteins, as assayed by SPR.

FIG. 5 summarizes the binding affinities of indicated trispecificbinding proteins against their cognate antigens (human CD3, CD28, andCD38) as measured by SPR.

FIG. 6A compares the apparent affinity of isatuximab antigen bindingdomain in human IgG1 format (2^(nd) sheet) or in a trispecific bindingprotein format with the isatuximab, anti-CD28, and anti-CD3 antigenbinding domains (format according to FIG. 3A; 1^(st) sheet) for bindinghuman (top) or cynomolgus monkey (bottom) CD38 polypeptides, as assayedby flow cytometry.

FIGS. 6B-6D compare the apparent affinities of trispecific bindingprotein CD38_(VH1)xCD28_(sup)xCD3_(mid),CD38_(VH1)xCD28_(cvn)xCD3_(mid), or monospecific anti-CD38 antibody mAb2for binding to cells expressing human or cynomolgus monkey CD38polypeptides, as assayed by flow cytometry. FIG. 6B shows the binding oftrispecific binding protein CD38_(VH1)xCD28_(sup)xCD3_(mid) to cellsexpressing human (top) or cynomolgus monkey (bottom) CD38 polypeptides.FIG. 6C shows the binding of trispecific binding proteinCD38_(VH1)xCD28_(cvn)xCD3_(mid) to cells expressing human (top) orcynomolgus monkey (bottom) CD38 polypeptides. FIG. 6D shows the bindingof monospecific anti-CD38 antibody mAb2 to cells expressing human (top)or cynomolgus monkey (bottom) CD38 polypeptides.

FIG. 6E compares the apparent affinities of trispecific binding proteinCD38_(HHY1370)xCD28_(sup)xCD3_(mid) or monospecific anti-CD38 antibodymAb6 for binding to cells expressing human (top) or cynomolgus monkey(bottom) CD38 polypeptides, as assayed by flow cytometry.

FIG. 6F summarizes the binding affinity of the indicatedanti-CD38xanti-CD28xanti-CD3 trispecific binding proteins for humanCD38, as measured by SPR or flow cytometry (FACS).

FIG. 6G shows the apparent affinity of trispecific binding proteinΔCD38_(VH1)xCD28_(sup)xCD3_(mid) lacking the anti-CD38 antigen bindingdomain for binding to cells expressing human (top) or cynomolgus monkey(bottom) CD38 polypeptides, as assayed by flow cytometry.

FIGS. 7A & 7B show the results of an ELISA assay determining the bindingaffinities of various anti-CD38 x CD28 x CD3 IgG4 trispecific bindingproteins, or control antibodies, to human and rhesus monkey CD3, CD28and CD38 polypeptides.

FIGS. 8A-8D show the results of antibody-mediated specific killing ofCD38⁺ cells by PBMCs from three different human donors using theindicated anti-CD38 x CD28 x CD3 trispecific binding proteins andcontrol antibodies. Representative results using multiple myeloma celllines RPMI8266 (FIG. 8A), NCI-H929 (FIG. 8B), KMS-26 (FIG. 8C), andKMS-11 cell lines (FIG. 8D) are shown, and EC50 values are provided inTable N. EC50 values obtained by using NCI-H929, KMS-26, and KMS-11cells are provided in Tables O-Q.

FIGS. 8E & 8F show the results of antibody-mediated specific killing ofCD38⁺ cells by PBMCs from two different donors using the indicatedanti-CD38 x CD28 x CD3 trispecific binding proteins with variant Fcregions and control antibodies. Representative results using CD38⁺KMS-11 (FIG. 8D) and U266 (FIG. 8E) cell lines are shown, and EC50values are provided in Tables Q2 and Q3.

FIGS. 9A, 9B, & 10 show the activation (CD69⁺) of human T cells treatedwith various anti-CD38 x CD28 x CD3 trispecific binding proteins orcontrol antibodies for 24 hours. FIG. 9A shows the activation (CD69⁺) ofhuman CD3⁺ T cells. FIG. 9B shows the activation (CD69⁺) of human CD3⁺CD4⁺ T cells. FIG. 10 shows the activation (CD69⁺) of human CD3⁺ CD8⁺ Tcells.

FIGS. 11A-11B show the results of in vitro cytokine release assessmentsof human PBMCs treated with the indicated anti-CD38 x CD28 x CD3trispecific binding proteins or control antibodies based on dried platemethod as described in Stebbings, R. et al. (2007) J. Immunol.179:3325-3331. FIG. 11A shows the results using 5 μg/mL of the indicatedantibodies. FIG. 11B shows the results using 25 ng/mL of the indicatedantibodies.

FIGS. 12A-12E show the in vivo activity of the anti-CD38_((VH1)) xCD28_((sup)) x CD3_((mid)) trispecific binding protein in the CD34+umbilical cord blood cells humanized NSG mouse model implanted withRPMI-8226 multiple myeloma cells. FIG. 12A shows the change in tumorvolume in mice treated with the indicated concentrations of theanti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid)) trispecific bindingprotein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG.12B shows the average tumor volume at day 18 in mice treated with theindicated concentrations of the anti-CD38_((VH1)) x CD28_((sup)) xCD3_((mid)) trispecific binding protein vs. mice treated with ananti-CD3/CD38 bispecific antibody. FIG. 12C shows the average terminaltumor weight in mice treated with the indicated concentrations of theanti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid)) trispecific bindingprotein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG.12D shows the average tumor growth curve over the length of theexperiment in mice treated with the indicated concentrations of theanti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid)) trispecific bindingprotein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG.12E shows the average change in body weight at multiple time points overthe length of the experiment of mice treated with the indicatedconcentrations of the anti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid))trispecific binding protein vs. mice treated with an anti-CD3/CD38bispecific antibody.

FIGS. 13A-13F show the in vivo activity of the anti-CD38_((VH1)) xCD28_((sup)) x CD3_((mid)) trispecific binding protein in the PBMCshumanized NSG mouse model implanted with RPMI-8226 multiple myelomacells. FIG. 13A shows the change in tumor volume in mice treated withthe indicated concentrations of the anti-CD38_((VH1)) x CD28_((sup)) xCD3_((mid)) trispecific binding protein vs. mice treated with ananti-CD3/CD38 bispecific antibody. FIG. 13B shows the tumor volume atday 4 in mice treated with the indicated concentrations of theanti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid)) trispecific bindingprotein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG.13C shows the tumor volume at day 21 in mice treated with the indicatedconcentrations of the anti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid))trispecific binding protein vs. mice treated with an anti-CD3/CD38bispecific antibody. FIG. 13D shows the average tumor volume at day 21in mice treated with the indicated concentrations of theanti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid)) trispecific bindingprotein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG.13E shows the average terminal tumor weight in mice treated with theindicated concentrations of the anti-CD38_((VH1)) x CD28_((sup)) xCD3_((mid)) trispecific binding protein vs. mice treated with ananti-CD3/CD38 bispecific antibody. FIG. 13F shows the average tumorvolume at multiple time points over the length of the experiment in micetreated with the indicated concentrations of the anti-CD38_((VH1)) xCD28_((sup)) x CD3_((mid)) trispecific binding protein vs. mice treatedwith an anti-CD3/CD38 bispecific antibody.

FIGS. 14A-14U show the results of a dose escalation study (0.5, 2.5,12.5 μg/kg) using the anti-CD38_((VH1))) x CD28_((sup)) x CD3_((mid)),the anti-CD38_((VH1)) x CD28_((cvn)) x CD3_((mid)), theanti-CD38_((hhy1370)) x CD28_((sup)) x CD3_((mid)), and theanti-CD38_((hhy1370)) x CD28_((cvn)) x CD3_((mid)) trispecific bindingproteins in non-human primates. FIG. 14A shows T cell activation (CD69⁺)of circulating CD3⁺ T cells after administration of different doses ofthe anti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid)) trispecific bindingprotein. FIG. 14B shows T cell activation (CD69⁺) of circulating CD3⁺ Tcells after administration of different doses of the anti-CD38_((VH1)) xCD28_((cvn)) x CD3_((mid)) trispecific binding protein. FIG. 14C shows Tcell activation (CD69⁺) of circulating CD3⁺ T cells after administrationof different doses of the anti-CD38_((hhy1370)) x CD28_((sup)) xCD3_((mid)) trispecific binding protein. FIG. 14D shows T cellactivation (CD69⁺) of circulating CD3⁺ T cells after administration ofdifferent doses of the anti-CD38_((hhy1370)) x CD28_((cvn)) xCD3_((mid)) trispecific binding protein. FIG. 14E shows the changes inpercentage of circulating CD4⁺ T cells after administration of theindicated doses of the anti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid))trispecific binding protein. FIG. 14F shows the changes in percentage ofcirculating CD8⁺ T cells after administration of the indicated doses ofthe anti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid)) trispecific bindingprotein. FIG. 14G shows the changes in percentage of circulating CD4⁺ Tcells after administration of the indicated doses of theanti-CD38_((VH1)) x CD28_((cvn)) x CD3_((mid)) trispecific bindingprotein. FIG. 14H shows the changes in percentage of circulating CD8⁺ Tcells after administration of the indicated doses of theanti-CD38_((VH1)) x CD28_((cvn)) x CD3_((mid)) trispecific bindingprotein. FIG. 14I shows the changes in percentage of circulating CD4⁺ Tcells after administration of the indicated doses of theanti-CD38_((hhy1370)) x CD28_((sup)) x CD3_((mid)) trispecific bindingprotein. FIG. 14J shows the changes in percentage of circulating CD8⁺ Tcells after administration of the indicated doses of theanti-CD38_((hhy1370)) x CD28_((sup)) x CD3_((mid)) trispecific bindingprotein. FIG. 14K shows the changes in percentage of circulating CD4⁺ Tcells after administration of the indicated doses of theanti-CD38_((hhy1370)) x CD28_((cvn)) x CD3_((mid)) trispecific bindingprotein. FIG. 14L shows the changes in percentage of circulating CD8⁺ Tcells after administration of the indicated doses of theanti-CD38_((hhy1370)) x CD28_((cvn)) x CD3_((mid)) trispecific bindingprotein. FIG. 14M shows the changes in total CD4⁺ T cells 6, 24, and 48hours after administration of 12.5 μg/kg of the indicated trispecificbinding proteins. FIG. 14N shows the changes in total NK cells 6, 24,and 48 hours after administration of 12.5 μg/kg of the indicatedtrispecific binding proteins. FIG. 14O shows the changes in total CD8⁺ Tcells 6, 24, and 48 hours after administration of 12.5 μg/kg of theindicated trispecific binding proteins. FIG. 14P shows the changes intotal B cells 6, 24, and 48 hours after administration of 12.5 μg/kg ofthe indicated trispecific binding proteins. FIG. 14Q shows the changesin cytokine levels 6 hours after administration of the three ascendingdoses (0.5, 2.5, 12.5 μg/kg) of the anti-CD38_((VH1)) x CD28_((sup)) xCD3_((mid)) trispecific binding protein (results from different testanimals labeled as “117065” and “117066”). FIG. 14R shows the changes incytokine levels 6 hours after administration of the three ascendingdoses (0.5, 2.5, 12.5 μg/kg) of the anti-CD38_((VH1)) x CD28_((cvn)) xCD3_((mid)) trispecific binding protein (results from different testanimals labeled as “117067” and “117068”). FIG. 14S shows the changes incytokine levels 6 hours after administration of the three ascendingdoses (0.5, 2.5, 12.5 μg/kg) of the anti-CD38_((hhy1370)) x CD28_((sup))x CD3_((mid)) trispecific binding protein (results from different testanimals labeled as “117069” and “117070”). FIG. 14T shows the changes incytokine levels 6 hours after administration of the three ascendingdoses (0.5, 2.5, 12.5 μg/kg) of the anti-CD38_((hhy1370)) x CD28_((cvn))x CD3_((mid)) trispecific binding protein (results from different testanimals labeled as “117071” and “117072”). FIG. 14U shows the changes incytokine levels 24 hours after administration of the three ascendingdoses (0.5, 2.5, 12.5 μg/kg) of the indicated trispecific bindingproteins (results shown from all test animals).

FIGS. 14V & 14W show that anti-CD38_((VH1)) x CD28_((sup)) x CD3_((mid))and anti-CD38_((VH1)) x CD28_((cvn)) x CD3_((mid)) trispecific bindingproteins induced depletion of T cells in vivo in non-human primate bloodat higher doses (6 hours post-dose).

FIGS. 14X & 14Y show that anti-CD38_((HHY1370)) x CD28_((sup)) xCD3_((mid)) and anti-CD38_((HHY1370)) x CD28_((cvn)) x CD3_((mid))trispecific binding proteins induced depletion of T cells in vivo innon-human primate blood at higher doses (6 hours post-dose).

FIGS. 14Z & 14AA show the amount of blood T cells in non-human primatesover time after administration of anti-CD38_((VH1)) x CD28_((sup)) xCD3_((mid)) or anti-CD38_((VH1)) x CD28_((cvn)) x CD3_((mid))trispecific binding proteins.

FIGS. 14AB & 14AC show the amount of blood T cells in non-human primatesover time after administration of anti-CD38_((HHY1370)) x CD28_((sup)) xCD3_((mid)) or anti-CD38_((HHY1370)) x CD28_((cvn)) x CD3_((mid))trispecific binding proteins.

FIGS. 14AD & 14AE show the amount of CD4+ T cells with trispecificbinding protein bound after administration of 100 g/kg dose in non-humanprimates.

FIGS. 14AF & 14AG show the amount of CD8+ T cells with trispecificbinding protein bound after administration of 100 μg/kg dose innon-human primates.

FIGS. 15A-15C show binding or lack thereof of various Fc variants tohuman Fc receptors FcγR I (FIG. 15A), FcγR IIa (FIG. 15B), and FcγRIIIb/c (FIG. 15C). Variants tested were human IgG1, human IgG4, andhuman IgG4 with FALA mutations.

FIG. 16 shows binding of human IgG4, with or without FALA mutations, toFcRn.

FIG. 17 summarizes PK parameters of the indicated trispecific bindingproteins (CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4,CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 FALA,CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG1 LALA P329A, andCD38_(HHY1370)xCD28_(sup)xCD3_(mid) IgG4 FALA) in NSG mice.

FIGS. 18A-18C show Fc/FcR interaction-mediated (non-specific) release ofIFN-γ (FIG. 18A), IL-2 (FIG. 18B), or TNF-α (FIG. 18C) by human PBMCsincubated with trispecific binding proteins having wild-type or FALAvariant Fc regions.

FIG. 18D shows in vitro activation of human PBMCs byCD38_(VH1)CD28_(sup)xCD3_(mid) and CD38_(HHY1370)xCD28_(sup)xCD3_(mid)trispecific binding proteins, as well as IgG1 and IgG4 Fc variantsthereof.

FIGS. 19A&19B show that induction of Bcl-xL in CD4+ (FIG. 19A) or CD8+(FIG. 19B) T cells by trispecific binding proteinCD38_(VH1)xCD28_(sup)xCD3_(mid) requires both CD3 and CD28 antigenbinding domains. Bar graph=mean and s.d. from 3 PBMC donors. *p=≤0.009.

FIGS. 19C&19D show that CD38_(VH1)xCD28xCD3 with IgG4 FALA variant Fcupregulates Bcl-xL in CD4+ (FIG. 19C) or CD8+ (FIG. 19D) T cells greaterthan a benchmark bispecific antibody. Bar graph=mean and s.d. from 3PBMC donors. *p=≤0.045

FIG. 19E shows that T cell activation by anti-CD38xanti-CD28xanti-CD3trispecific binding proteins, as assayed by IL-2 expression in a JurkatT cell reporter line, is dependent upon the anti-CD3 antigen bindingdomain.

FIG. 19F shows the release of cytokines TNF, IFNg, IL-2, IL-6, and IL-10by CD38_(VH1)xCD28_(sup)xCD3_(mid) trispecific binding proteins, ascompared to binding proteins with mutated anti-CD28, anti-CD38, oranti-CD28, anti-CD38, and anti-CD3, as well as the benchmark.

FIG. 19G shows proliferation of T cells activated byanti-CD38xanti-CD28xanti-CD3 trispecific binding protein with IgG4 FALAvariant Fc, benchmark anti-CD38xanti-CD3 bispecific antibody, or isotypecontrol (trispecific binding protein with IgG4 FALA variant Fc havingmutated binding domains).

FIG. 20 shows proliferation of T cells activated byanti-CD38xanti-CD28xanti-CD3 trispecific binding protein with IgG4 FALAvariant Fc, anti-CD38xanti-CD28xanti-CD3 trispecific binding proteinswith IgG4 FALA variant Fc and a mutation in the CD38, CD28, or CD3antigen binding domain, or isotype control (trispecific binding proteinwith IgG4 FALA variant Fc having three mutated binding domains).

FIG. 21 shows in vivo anti-tumor activity ofCD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 FALA trispecific binding proteinadministered at the indicated doses in an NCI-H929-Luc disseminatedtumor model in PBMC humanized NSG mice.

FIG. 22 shows in vivo anti-tumor activity ofCD38_(HHY1370)xCD28_(sup)xCD3_(mid) IgG4 FALA trispecific bindingprotein administered at the indicated doses in an NCI-H929-Lucdisseminated tumor model in PBMC humanized NSG mice.

FIGS. 23A & 23B show potent in vitro tumor killing activity ofNCI-929-Luc cells with CD38_(VH1)xCD28_(sup)xCD3_(mid) andCD38_(HHY1370)xCD28_(sup)xCD3_(mid) trispecific binding proteins, andbenchmark anti-CD38xanti-CD3 bispecific antibody using the human PBMCsused in the in vivo study. Human PBMCs from two donor humanized NSG micewere used after 24 h. incubation with an effector:PBMC ratio of 10:1.

FIG. 23C shows superior in vivo anti-tumor activity ofCD38_(VH1)xCD28_(sup)xCD3_(mid) and CD38_(hhy1370)xCD28_(sup)xCD3_(mid)trispecific binding proteins, as compared to benchmarkanti-CD38xanti-CD3 bispecific antibody, administered at the indicateddoses in an NCI-H929-Luc disseminated tumor model in PBMC humanized NSGmice. Binding proteins were administered by weekly intraperitoneal (IP)injection at 30 μg/kg.

FIG. 24A shows the results of a luciferase reporter assay usingGloResponse™ IL2-luc2P Jurkat Cells (Promega) after stimulation byCD38_(VH1)/CD28_(sup)xCD3_(mid) and its single binding site KO andtriple KO mutants at 10 nM concentration.

FIG. 24B shows the optimization of anti-CD3xCD28 CODV-Fab antibody. Theoptimal configuration of the α-CD3 and α-CD28 in alternative positionsof the CODV bispecific Fab was evaluated by cytokine release assaysusing human PBMCs in vitro. Distal-CD28 x proximal CD3 was identified asoptimal positioning based on the secretion of IFN-γ and IL-2 insupernatant after 24 hours.

FIG. 25 shows that CD38_(VH1)/CD28_(sup)xCD3_(mid) induced upregulationof Bcl-2 family member Bcl-xL in primary T cells is CD28 dependent.

FIG. 26 shows that anti-CD28 in the trispecific Ab provided secondarysignaling essential for supporting primary T cell proliferation invitro.

FIG. 27 shows the configuration of the trispecific antibody, color-codedby parental antibody (left). Dark shades (purple or green) denote heavychain peptides; light shades denote light chain peptides. Also shown isa structure model of the CD38_(VH1)/CD28_(sup)xCD3_(mid) trispecific Abbased on crystal structures of anti-CD38 VH1 Fab andCD28_(sup)/CD3_(mid) CODV Fab (right).

FIGS. 28A & 28B show that multiple myeloma (MM) cells with high(RPMI-8226; FIG. 28A) and low (KMS-11; FIG. 28B) CD38 surface expressionwere lysed efficiently by human PBMCs (E:T=10:1) incubated with variousconcentrations of the trispecific Ab. Contribution to the killingactivity by each binding site was demonstrated by binding site KOmutations, as indicated.

FIGS. 29A-29C show that the anti-CD28_(sup)KO mutant of the trispecificAb exhibited markedly reduced anti-tumor activity against CD38_(high),CD38_(mid) and CD38_(low) MM cells in vitro. Shown are assays usingRPMI-8226 (FIG. 29A), U266 (FIG. 29B), or KMS-11 (FIG. 29C) cells.

FIG. 30 shows that reduction of tumor burden inCD38_(VH1)/CD28_(sup)xCD3_(mid)_FALA trispecific antibody treatmentgroups was dose-dependent and statistically different in a disseminatedhuman multiple myeloma cell line model using an NSG mouse reconstitutedwith in vitro amplified human primary T cells.

FIGS. 31A & 31B show vital microscopy analysis of myeloma cell cytolysisby the CD38 trispecific Ab in vitro in the presence of primary human Tcells. Time lapse photography of microscopic images was performed usinga negative control (triple KO trispecific; FIG. 31A) or theCD38/CD28xCD3 trispecific Ab (FIG. 31B) using human PBMCs incubated withRPMI-8226 myeloma cell line labeled with CellTracker™ deep red dye.Images presented were collected after 24 hour incubation. Scale bar: 50μm.

FIG. 32 shows alternative mutations in the Fc region of IgG4 preparedfor analysis in Fc receptor binding assays. Shown are SEQ ID NOs:111-116(top to bottom, respectively).

FIG. 33 shows the results of SPR assays to measure the affinity of thespecified IgG4 Fc variants to the indicated human Fc receptors.

FIG. 34 shows that a CD38 trispecific binding protein with minimal FcRbinding reduced non-specific cytokine release by human PBMCs in vitro.Different FcR inactivating mutations (as indicated) were analyzed fortheir proinflammatory effects in the human IgG4 isotype. Human PBMCswere incubated in media (Unstimulated) or in the presence of the myelomacells, RPMI-8226 (Stimulated), and bars indicate supernatant IFN-γlevels measured by ELISA.

FIG. 35 shows that a CD38 trispecific binding protein with minimal FcRbinding lysed human multiple myeloma cells with different CD38expression levels. Cytolysis of myeloma cells with IgG4 or the indicatedFc mutations was assessed in vitro using human PBMCs with the indicatedtumor targets.

FIG. 36 shows a comparison of in vitro cytolytic activity of thetrispecific anti-CD38/CD28/CD3 Ab and the anti-CD38 antibody daratumumabmeasured using human PBMC as effector cells against cell linesRPMI-8226, U266, and KMS-11 (E:T=10:1).

FIGS. 37A-37D show the characterization of in vitro T cell subsetexpansion in response to CD38/CD3xCD28. Evaluation of T cell subsetexpansion was performed by coating wells with 350 ng/well of the CD38trispecific Ab in the absence of exogenous cytokines. T cell populationswere measured at indicated time points. The triple mutant trispecific abwas used as negative control. Flow cytometry was used to determinecentral and effector memory CD4 T cells (FIG. 37A), T helper cells (FIG.37B), central and effector memory CD8 T cells (FIG. 37C), and CMVpp65-specific CD8 cells (FIG. 37D) as described in Example 12. Analysisof CMV-specific pp65 effector cells was performed by pentamer stainingof PBMCs from HLA-A2 CMV+ donors treated with the CD38 trispecific orthe triple negative control.

FIG. 38 shows the contribution of CD28 expression on target cells tosusceptibility to cytolysis by CD38/CD3xCD28. CD28 was knocked out inKMS-11 cells using CRISPR/Cas 9 gene targeting and used as cytolytictargets in vitro. Compared to parental KMS-11, CD38 expression waspreserved while CD28 was eliminated, as demonstrated by flow cytometry(upper panel, KMS-11 vs. KMS-11 (CD28KO). Cytolysis of the CD28 KO cellswas examined with the WT or CD28 null trispecific (lower panel;trispecific vs. trispecific (CD28KO)).

FIG. 39 shows the cytolytic activity of the CD38/CD28xCD3 trispecificFALA mutant Ab against indicated CD38⁺CD28⁻ lines, including acutemyelocytic leukemia (AML(KG-1)), a B cell lymphoma (OCI-Ly19), acute Tlymphocytic leukemia (ALL (KOPN8)), and chronic lymphocytic lymphoma(CLL(Z-138)).

FIG. 40 shows that in vitro activation of human PBMC by α-CD28superagonist requires bivalency of the antibody.

DETAILED DESCRIPTION

The disclosure provides binding protein comprising at least one antigenbinding site that binds a CD38 polypeptide.

I. General Definitions

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings. Unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

It is understood that aspects and embodiments of the present disclosuredescribed herein include “comprising,” “consisting,” and “consistingessentially of” aspects and embodiments.

The term “polynucleotide” as used herein refers to single-stranded ordouble-stranded nucleic acid polymers of at least 10 nucleotides inlength. In certain embodiments, the nucleotides comprising thepolynucleotide can be ribonucleotides or deoxyribonucleotides or amodified form of either type of nucleotide. Such modifications includebase modifications such as bromuridine, ribose modifications such asarabinoside and 2′,3′-dideoxyribose, and internucleotide linkagemodifications such as phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phoshoraniladate and phosphoroamidate. The term “polynucleotide”specifically includes single-stranded and double-stranded forms of DNA.

An “isolated polynucleotide” is a polynucleotide of genomic, cDNA, orsynthetic origin or some combination thereof, which: (1) is notassociated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, (2) is linked to apolynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

An “isolated polypeptide” is one that: (1) is free of at least someother polypeptides with which it would normally be found, (2) isessentially free of other polypeptides from the same source, e.g., fromthe same species, (3) is expressed by a cell from a different species,(4) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates, or other materials with which itis associated in nature, (5) is not associated (by covalent ornoncovalent interaction) with portions of a polypeptide with which the“isolated polypeptide” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated polypeptide can be encoded by genomic DNA,cDNA, mRNA or other RNA, of synthetic origin, or any combinationthereof. Preferably, the isolated polypeptide is substantially free frompolypeptides or other contaminants that are found in its naturalenvironment that would interfere with its use (therapeutic, diagnostic,prophylactic, research or otherwise).

Naturally occurring antibodies typically comprise a tetramer. Each suchtetramer is typically composed of two identical pairs of polypeptidechains, each pair having one full-length “light” chain (typically havinga molecular weight of about 25 kDa) and one full-length “heavy” chain(typically having a molecular weight of about 50-70 kDa). The terms“heavy chain” and “light chain” as used herein refer to anyimmunoglobulin polypeptide having sufficient variable domain sequence toconfer specificity for a target antigen. The amino-terminal portion ofeach light and heavy chain typically includes a variable domain of about100 to 110 or more amino acids that typically is responsible for antigenrecognition. The carboxy-terminal portion of each chain typicallydefines a constant domain responsible for effector function. Thus, in anaturally occurring antibody, a full-length heavy chain immunoglobulinpolypeptide includes a variable domain (V_(H)) and three constantdomains (C_(H1), C_(H2), and C_(H3)), wherein the V_(H) domain is at theamino-terminus of the polypeptide and the C_(H3) domain is at thecarboxyl-terminus, and a full-length light chain immunoglobulinpolypeptide includes a variable domain (V_(L)) and a constant domain(C_(L)), wherein the V_(L) domain is at the amino-terminus of thepolypeptide and the C_(L) domain is at the carboxyl-terminus.

Human light chains are typically classified as kappa and lambda lightchains, and human heavy chains are typically classified as mu, delta,gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD,IgG, IgA, and IgE, respectively. IgG has several subclasses, including,but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclassesincluding, but not limited to, IgM1 and IgM2. IgA is similarlysubdivided into subclasses including, but not limited to, IgA1 and IgA2.Within full-length light and heavy chains, the variable and constantdomains typically are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See, e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., RavenPress, 2nd ed., 1989), which is incorporated by reference in itsentirety for all purposes. The variable regions of each light/heavychain pair typically form an antigen binding site. The variable domainsof naturally occurring antibodies typically exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair typically are alignedby the framework regions, which may enable binding to a specificepitope. From the amino-terminus to the carboxyl-terminus, both lightand heavy chain variable domains typically comprise the domains FR1,CDR1, FR2, CDR2, FR3, CDR3, and FR4.

The term “CDR set” refers to a group of three CDRs that occur in asingle variable region capable of binding the antigen. The exactboundaries of these CDRs have been defined differently according todifferent systems. The system described by Kabat (Kabat et al.,SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17; Chothia etal., 1989, Nature 342: 877-83) found that certain sub-portions withinKabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2, and L3 or H1, H2, and H3where the “L” and the “H” designates the light chain and the heavy chainregions, respectively. These regions may be referred to as Chothia CDRs,which have boundaries that overlap with Kabat CDRs. Other boundariesdefining CDRs overlapping with the Kabat CDRs have been described byPadlan, 1995, FASEB J. 9: 133-39; MacCallum, 1996, J. Mol. Biol. 262(5):732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27: 55-77. Still otherCDR boundary definitions may not strictly follow one of the hereinsystems, but will nonetheless overlap with the Kabat CDRs, although theymay be shortened or lengthened in light of prediction or experimentalfindings that particular residues or groups of residues or even entireCDRs do not significantly impact antigen binding. The methods usedherein may utilize CDRs defined according to any of these systems,although certain embodiments use Kabat or Chothia defined CDRs.Identification of predicted CDRs using the amino acid sequence is wellknown in the field, such as in Martin, A. C. “Protein sequence andstructure analysis of antibody variable domains,” In AntibodyEngineering, Vol. 2. Kontermann R., Dübel S., eds. Springer-Verlag,Berlin, p. 33-51 (2010). The amino acid sequence of the heavy and/orlight chain variable domain may be also inspected to identify thesequences of the CDRs by other conventional methods, e.g., by comparisonto known amino acid sequences of other heavy and light chain variableregions to determine the regions of sequence hypervariability. Thenumbered sequences may be aligned by eye, or by employing an alignmentprogram such as one of the CLUSTAL suite of programs, as described inThompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular models areconventionally used to correctly delineate framework and CDR regions andthus correct the sequence-based assignments.

In some embodiments, CDR/FR definition in an immunoglobulin light orheavy chain is to be determined based on IMGT definition (Lefranc et al.Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

The term “Fc” as used herein refers to a molecule comprising thesequence of a non-antigen-binding fragment resulting from digestion ofan antibody or produced by other means, whether in monomeric ormultimeric form, and can contain the hinge region. The originalimmunoglobulin source of the native Fc is preferably of human origin andcan be any of the immunoglobulins. Fc molecules are made up of monomericpolypeptides that can be linked into dimeric or multimeric forms bycovalent (i.e., disulfide bonds) and non-covalent association. Thenumber of intermolecular disulfide bonds between monomeric subunits ofnative Fc molecules ranges from 1 to 4 depending on class (e.g., IgG,IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, andIgG4). One example of a Fc is a disulfide-bonded dimer resulting frompapain digestion of an IgG. The term “native Fc” as used herein isgeneric to the monomeric, dimeric, and multimeric forms.

A F(ab) fragment typically includes one light chain and the V_(H) andC_(H1) domains of one heavy chain, wherein the V_(H)-C_(H1) heavy chainportion of the F(ab) fragment cannot form a disulfide bond with anotherheavy chain polypeptide. As used herein, a F(ab) fragment can alsoinclude one light chain containing two variable domains separated by anamino acid linker and one heavy chain containing two variable domainsseparated by an amino acid linker and a C_(H1) domain.

A F(ab′) fragment typically includes one light chain and a portion ofone heavy chain that contains more of the constant region (between theC_(H1) and C_(H2) domains), such that an interchain disulfide bond canbe formed between two heavy chains to form a F(ab′)₂ molecule.

The term “binding protein” as used herein refers to a non-naturallyoccurring (or recombinant or engineered) molecule that specificallybinds to at least one target antigen, e.g., a CD38 polypeptide of thepresent disclosure

A “recombinant” molecule is one that has been prepared, expressed,created, or isolated by recombinant means.

One embodiment of the disclosure provides binding proteins havingbiological and immunological specificity to between one and three targetantigens. Another embodiment of the disclosure provides nucleic acidmolecules comprising nucleotide sequences encoding polypeptide chainsthat form such binding proteins. Another embodiment of the disclosureprovides expression vectors comprising nucleic acid molecules comprisingnucleotide sequences encoding polypeptide chains that form such bindingproteins. Yet another embodiment of the disclosure provides host cellsthat express such binding proteins (i.e., comprising nucleic acidmolecules or vectors encoding polypeptide chains that form such bindingproteins).

The term “swapability” as used herein refers to the interchangeabilityof variable domains within the binding protein format and with retentionof folding and ultimate binding affinity. “Full swapability” refers tothe ability to swap the order of both V_(H1) and V_(H2) domains, andtherefore the order of V_(L1) and V_(L2) domains, in the polypeptidechain of formula I or the polypeptide chain of formula II (i.e., toreverse the order) while maintaining full functionality of the bindingprotein as evidenced by the retention of binding affinity. Furthermore,it should be noted that the designations V_(H) and V_(L) refer only tothe domain's location on a particular protein chain in the final format.For example, V_(H1) and V_(H2) could be derived from V_(L1) and V_(L2)domains in parent antibodies and placed into the V_(H1) and V_(H2)positions in the binding protein. Likewise, V_(L1) and V_(L2) could bederived from V_(H1) and V_(H2) domains in parent antibodies and placedin the V_(H1) and V_(H2) positions in the binding protein. Thus, theV_(H) and V_(L) designations refer to the present location and not theoriginal location in a parent antibody. V_(H) and V_(L) domains aretherefore “swappable.”

The term “antigen” or “target antigen” or “antigen target” as usedherein refers to a molecule or a portion of a molecule that is capableof being bound by a binding protein, and additionally is capable ofbeing used in an animal to produce antibodies capable of binding to anepitope of that antigen. A target antigen may have one or more epitopes.With respect to each target antigen recognized by a binding protein, thebinding protein is capable of competing with an intact antibody thatrecognizes the target antigen.

“CD38” is cluster of differentiation 38 polypeptide and is aglycoprotein found on the surface of many immune cells. In someembodiments, a binding protein of the present disclosure binds theextracellular domain of one or more CD38 polypeptide. Exemplary CD38extracellular domain polypeptide sequences include, but are not limitedto, the extracellular domain of human CD38 (e.g., as represented by SEQID NO:1) and the extracellular domain of cynomolgus monkey CD38 (e.g.,as represented by SEQ ID NO:30).

The term “T-cell engager” refers to binding proteins directed to ahost's immune system, more specifically the T cells' cytotoxic activityas well as directed to a tumor target protein.

The term “monospecific binding protein” refers to a binding protein thatspecifically binds to one antigen target.

The term “monovalent binding protein” refers to a binding protein thathas one antigen binding site.

The term “bispecific binding protein” refers to a binding protein thatspecifically binds to two different antigen targets. In someembodiments, a bispecific binding protein binds to two differentantigens. In some embodiments, a bispecific binding protein binds to twodifferent epitopes on the same antigen.

The term “bivalent binding protein” refers to a binding protein that hastwo binding sites.

The term “trispecific binding protein” refers to a binding protein thatspecifically binds to three different antigen targets. In someembodiments, a trispecific binding protein binds to three differentantigens. In some embodiments, a trispecific binding protein binds toone, two, or three different epitopes on the same antigen.

The term “trivalent binding protein” refers to a binding protein thathas three binding sites. In particular embodiments the trivalent bindingprotein can bind to one antigen target. In other embodiments, thetrivalent binding protein can bind to two antigen targets. In otherembodiments, the trivalent binding protein can bind to three antigentargets.

An “isolated” binding protein is one that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the bindingprotein, and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In some embodiments, the binding protein willbe purified: (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated bindingproteins include the binding protein in situ within recombinant cellssince at least one component of the binding protein's naturalenvironment will not be present.

The terms “substantially pure” or “substantially purified” as usedherein refer to a compound or species that is the predominant speciespresent (i.e., on a molar basis it is more abundant than any otherindividual species in the composition). In some embodiments, asubstantially purified fraction is a composition wherein the speciescomprises at least about 50% (on a molar basis) of all macromolecularspecies present. In other embodiments, a substantially pure compositionwill comprise more than about 80%, 85%, 90%, 95%, or 99% of allmacromolar species present in the composition. In still otherembodiments, the species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specifically binding to an immunoglobulin orT-cell receptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody or binding protein. Incertain embodiments, a binding protein is said to specifically bind anantigen when it preferentially recognizes its target antigen in acomplex mixture of proteins and/or macromolecules. In some embodiments,a binding protein is said to specifically bind an antigen when theequilibrium dissociation constant is ≤10⁻⁸ M, more preferably when theequilibrium dissociation constant is ≤10⁻⁹ M, and most preferably whenthe dissociation constant is ≤10⁻¹⁰ M.

The dissociation constant (K_(D)) of a binding protein can bedetermined, for example, by surface plasmon resonance. Generally,surface plasmon resonance analysis measures real-time bindinginteractions between ligand (a target antigen on a biosensor matrix) andanalyte (a binding protein in solution) by surface plasmon resonance(SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, N.J.).Surface plasmon analysis can also be performed by immobilizing theanalyte (binding protein on a biosensor matrix) and presenting theligand (target antigen). The term “K_(D),” as used herein refers to thedissociation constant of the interaction between a particular bindingprotein and a target antigen.

The term “binds to” as used herein in reference to a binding proteinrefers to the ability of a binding protein or an antigen-bindingfragment thereof to bind to an antigen containing an epitope with an Kdof at least about 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M,1×10⁻¹¹ M, 1×10⁻¹² M, or more, and/or to bind to an epitope with anaffinity that is at least two-fold greater than its affinity for anonspecific antigen. In some embodiments, a binding protein of thepresent disclosure binds to two or more antigens, e.g., a human and acynomologus monkey CD38 polypeptide.

In some embodiments, an antigen binding domain and/or binding protein ofthe present disclosure “cross reacts” with human and cynomolgus monkeyCD38 polypeptides, e.g., CD38 extracellular domains, such as SEQ ID NO:1(human CD38 isoform A), SEQ ID NO:105 (human CD38 isoform E) and SEQ IDNO:30 (cynomolgus monkey CD38). A binding protein binding to antigen 1(Ag1) is “cross-reactive” to antigen 2 (Ag2) when the EC50s are in asimilar range for both antigens. In the present application, a bindingprotein binding to Ag1 is cross-reactive to Ag2 when the ratio ofaffinity of Ag2 to affinity of Ag1 is equal or less than 10 (forinstance 5, 2, 1 or 0.5), affinities being measured with the same methodfor both antigens.

A binding protein binding to Ag1 is “notsignificantly cross-reactive” toAg2 when the affinities are very different for the two antigens.Affinity for Ag2 may not be measurable if the binding response is toolow. In the present application, a binding protein binding to Ag1 is notsignificantly cross-reactive to Ag2, when the binding response of thebinding protein to Ag2 is less than 5% of the binding response of thesame binding protein to Ag1 in the same experimental setting and at thesame antibody concentration. In practice, the binding proteinconcentration used can be the EC50 or the concentration required toreach the saturation plateau obtained with Ag1.

The term “linker” as used herein refers to one or more amino acidresidues inserted between immunoglobulin domains to provide sufficientmobility for the domains of the light and heavy chains to fold intocross over dual variable region immunoglobulins. A linker is inserted atthe transition between variable domains or between variable and constantdomains, respectively, at the sequence level. The transition betweendomains can be identified because the approximate size of theimmunoglobulin domains are well understood. The precise location of adomain transition can be determined by locating peptide stretches thatdo not form secondary structural elements such as beta-sheets oralpha-helices as demonstrated by experimental data or as can be assumedby techniques of modeling or secondary structure prediction. The linkersdescribed herein are referred to as L₁, which is located on the lightchain between the C-terminus of the V_(L1) and the N-terminus of theV_(L1) domain; and L₂, which is located on the light chain between theC-terminus of the V_(L1) and the N-terminus of the C_(L) domain. Theheavy chain linkers are known as L₃, which is located between theC-terminus of the V_(H1) and the N-terminus of the V_(H2) domain; andL₄, which is located between the C-terminus of the V_(H2) and theN-terminus of the C_(H1) domain.

The term “vector” as used herein refers to any molecule (e.g., nucleicacid, plasmid, or virus) that is used to transfer coding information toa host cell. The term “vector” includes a nucleic acid molecule that iscapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid,” which refers to a circulardouble-stranded DNA molecule into which additional DNA segments may beinserted. Another type of vector is a viral vector, wherein additionalDNA segments may be inserted into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell and thereby arereplicated along with the host genome. In addition, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. The terms “plasmid” and “vector” may be usedinterchangeably herein, as a plasmid is the most commonly used form ofvector. However, the disclosure is intended to include other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses, and adeno-associated viruses), which serveequivalent functions.

The phrase “recombinant host cell” (or “host cell”) as used hereinrefers to a cell into which a recombinant expression vector has beenintroduced. A recombinant host cell or host cell is intended to refernot only to the particular subject cell, but also to the progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but suchcells are still included within the scope of the term “host cell” asused herein. A wide variety of host cell expression systems can be usedto express the binding proteins, including bacterial, yeast,baculoviral, and mammalian expression systems (as well as phage displayexpression systems). An example of a suitable bacterial expressionvector is pUC19. To express a binding protein recombinantly, a host cellis transformed or transfected with one or more recombinant expressionvectors carrying DNA fragments encoding the polypeptide chains of thebinding protein such that the polypeptide chains are expressed in thehost cell and, preferably, secreted into the medium in which the hostcells are cultured, from which medium the binding protein can berecovered.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransformation, the transforming DNA may recombine with that of the cellby physically integrating into a chromosome of the cell, or may bemaintained transiently as an episomal element without being replicated,or may replicate independently as a plasmid. A cell is considered tohave been stably transformed when the DNA is replicated with thedivision of the cell. The term “transfection” as used herein refers tothe uptake of foreign or exogenous DNA by a cell, and a cell has been“transfected” when the exogenous DNA has been introduced inside the cellmembrane. A number of transfection techniques are well known in the art.Such techniques can be used to introduce one or more exogenous DNAmolecules into suitable host cells.

The term “naturally occurring” as used herein and applied to an objectrefers to the fact that the object can be found in nature and has notbeen manipulated by man. For example, a polynucleotide or polypeptidethat is present in an organism (including viruses) that can be isolatedfrom a source in nature and that has not been intentionally modified byman is naturally-occurring. Similarly, “non-naturally occurring” as usedherein refers to an object that is not found in nature or that has beenstructurally modified or synthesized by man.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. Stereoisomers (e.g., D-aminoacids) of the twenty conventional amino acids; unnatural amino acids andanalogs such as α-, α-disubstituted amino acids, N-alkyl amino acids,lactic acid, and other unconventional amino acids may also be suitablecomponents for the polypeptide chains of the binding proteins. Examplesof unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxyl-terminal direction, in accordancewith standard usage and convention.

Naturally occurring residues may be divided into classes based on commonside chain properties:

(1) hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

(2) polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

(3) aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

(4) aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;

(5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(6) acidic: Asp, Glu;

(7) basic: His, Lys, Arg;

(8) residues that influence chain orientation: Gly, Pro;

(9) aromatic: His, Trp, Tyr, Phe; and

(10) aromatic hydrophobic: Phe, Trp, Tyr.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class.

A skilled artisan will be able to determine suitable variants of thepolypeptide chains of the binding proteins using well-known techniques.For example, one skilled in the art may identify suitable areas of apolypeptide chain that may be changed without destroying activity bytargeting regions not believed to be important for activity.Alternatively, one skilled in the art can identify residues and portionsof the molecules that are conserved among similar polypeptides. Inaddition, even areas that may be important for biological activity orfor structure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

The term “patient” as used herein includes human and animal subjects(e.g., mammals, such as dogs, pigs, horses, cats, cows, etc.).

The terms “treatment” or “treat” as used herein refer to boththerapeutic treatment and prophylactic or preventative measures. Thosein need of treatment include those having a disorder as well as thoseprone to have the disorder or those in which the disorder is to beprevented. In particular embodiments, binding proteins can be used totreat humans with cancer, or humans susceptible to cancer, or amelioratecancer in a human subject. The binding proteins can also be used toprevent cancer in a human patient. In particular embodiments, the canceris multiple myeloma, acute lymphoblastic leukemia, chronic lymphocyticleukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+breast cancer, prostate cancer, germinal center B-cell lympohoma orB-cell acute lymphoblastic leukemia.

The terms “pharmaceutical composition” or “therapeutic composition” asused herein refer to a compound or composition capable of inducing adesired therapeutic effect when properly administered to a patient.

The term “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier” as used herein refers to one or more formulationmaterials suitable for accomplishing or enhancing the delivery of abinding protein.

The terms “effective amount” and “therapeutically effective amount” whenused in reference to a pharmaceutical composition comprising one or morebinding proteins refer to an amount or dosage sufficient to produce adesired therapeutic result. More specifically, a therapeuticallyeffective amount is an amount of a binding protein sufficient toinhibit, for some period of time, one or more of the clinically definedpathological processes associated with the condition being treated. Theeffective amount may vary depending on the specific binding protein thatis being used, and also depends on a variety of factors and conditionsrelated to the patient being treated and the severity of the disorder.For example, if the binding protein is to be administered in vivo,factors such as the age, weight, and health of the patient as well asdose response curves and toxicity data obtained in preclinical animalwork would be among those factors considered. The determination of aneffective amount or therapeutically effective amount of a givenpharmaceutical composition is well within the ability of those skilledin the art.

One embodiment of the disclosure provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a binding protein.

II. Anti-CD38 Binding Proteins

Certain aspects of the present disclosure relate to binding proteinsthat comprise an antigen binding site that binds a CD38 polypeptide(e.g., human and cynomolgus monkey CD38 polypeptides). In someembodiments, the binding proteins are monospecific and/or monovalent,bispecific and/or bivalent, trispecific and/or trivalent, ormultispecific and/or multivalent.

A variety of features of exemplary monospecific, bispecific, ortrispecific binding proteins are described herein. For example, in someembodiments, a binding protein or antigen-binding fragment thereofcross-reacts with human CD38 (e.g., a human CD38 isoform A and/orisoform E polypeptide) and cynomolgus monkey CD38. In some embodiments,a binding protein induces apoptosis of a CD38+ cell. In someembodiments, a binding protein recruits a T cell to a CD38+ cell andoptionally activates the T cell (e.g., though TCR stimulation and/orcostimulation).

In some embodiments, the binding proteins comprise an antigen bindingsite comprising: an antibody heavy chain variable (VH) domain comprisinga CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ IDNO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising theamino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ IDNO:38), and a CDR-H3 sequence comprising the amino acid sequence ofARTGGLRRAYFTY (SEQ ID NO:33); or an antibody light chain variable (VL)domain comprising a CDR-L1 sequence comprising the amino acid sequenceof ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding proteinscomprise an antigen binding site comprising: an antibody heavy chainvariable (VH) domain comprising a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), aCDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ IDNO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising theamino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); or an antibodylight chain variable (VL) domain comprising a CDR-L1 sequence comprisingthe amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS(SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprisingthe amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In someembodiments, the binding proteins comprise 1, 2, 3, 4, 5, or 6 CDRs froman antibody VH and/or VL domain sequence of mAb1, mAb2, mAb3, mAb4,mAb5, mAb6, mAb2xCD28supxCD3mid IgG4 FALA, mAb2xCD28supxCD3mid IgG1LALAP329A, mAb2xCD28supxCD3mid IgG1 NNSA, mAb6xCD28supxCD3mid IgG4 FALA,mAb6xCD28supxCD3mid IgG1LALA P329A, or mAb6xCD28supxCD3mid IgG1 NNSA, asshown in Table G, H, or I.

In some embodiments, the binding proteins comprise an antigen bindingsite comprising: an antibody heavy chain variable (VH) domain comprisinga CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ IDNO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising theamino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ IDNO:38), and a CDR-H3 sequence comprising the amino acid sequence ofARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable (VL)domain comprising a CDR-L1 sequence comprising the amino acid sequenceof ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36).

In some embodiments, the binding proteins comprise an antigen bindingsite comprising: an antibody heavy chain variable (VH) domain comprisinga CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ IDNO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT(SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequenceof ARTGGLRRAYFTY (SEQ ID NO:33); or an antibody light chain variable(VL) domain comprising a CDR-L1 sequence comprising the amino acidsequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising theamino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In someembodiments, the binding proteins comprise an antigen binding sitecomprising: an antibody heavy chain variable (VH) domain comprising aCDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ IDNO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT(SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequenceof ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable(VL) domain comprising a CDR-L1 sequence comprising the amino acidsequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising theamino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In otherembodiments, the binding proteins comprise an antigen binding sitecomprising: an antibody heavy chain variable (VH) domain comprising aCDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ IDNO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT(SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequenceof ARTGGLRRAYFTY (SEQ ID NO:33); or an antibody light chain variable(VL) domain comprising a CDR-L1 sequence comprising the amino acidsequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising theamino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In someembodiments, the binding proteins comprise an antigen binding sitecomprising: an antibody heavy chain variable (VH) domain comprising aCDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ IDNO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT(SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequenceof ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable(VL) domain comprising a CDR-L1 sequence comprising the amino acidsequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising theamino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).

In some embodiments, the VH domain comprises the sequence, fromN-terminus to C-terminus, FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; whereFR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86),QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87), or QVQLVQSGAEVVKPGASVKMSCKAS(SEQ ID NO:88); where FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO:91); where FR3 comprises thesequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) orNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and where FR4comprises the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments,the VL domain comprises the sequence, from N-terminus to C-terminus,FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4; where FR1 comprises the sequenceDIVLTQSPATLSLSPGERATISCRAS (SEQ ID NO:97); where FR2 comprises thesequence MHWYQQKPGQPPRLLIY (SEQ ID NO:99); where FR3 comprises thesequence SRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYC (SEQ ID NO:101); and whereFR4 comprises the sequence FGGGTKLEIK (SEQ ID NO:103).

In some embodiments, the VH domain comprises an amino acid sequence thatis at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acid sequence of SEQ ID NO:5; and/or the VLdomain comprises an amino acid sequence that is at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to theamino acid sequence of SEQ ID NO:6. In some embodiments, the VH domaincomprises an amino acid sequence that is at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to the aminoacid sequence of SEQ ID NO:17; and/or the VL domain comprises an aminoacid sequence that is at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical to the amino acid sequence of SEQID NO:18. In some embodiments, the VH domain comprises an amino acidsequence that is at least 85%, at least 86%, at least 87%, at least 88%,at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to the amino acid sequence of SEQ ID NO:21;and/or the VL domain comprises an amino acid sequence that is at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO:18. In someembodiments, the VH domain comprises an amino acid sequence that is atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acid sequence of SEQ ID NO:23; and/or the VLdomain comprises an amino acid sequence that is at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to theamino acid sequence of SEQ ID NO:18. In some embodiments, the VH domaincomprises an amino acid sequence that is at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to the aminoacid sequence of SEQ ID NO:13; and/or the VL domain comprises an aminoacid sequence that is at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical to the amino acid sequence of SEQID NO:14.

In some embodiments, the VH domain comprises an amino acid sequence thatis at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acid sequence of SEQ ID NO:5; and the VLdomain comprises an amino acid sequence that is at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to theamino acid sequence of SEQ ID NO:6. In some embodiments, the VH domaincomprises an amino acid sequence that is at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to the aminoacid sequence of SEQ ID NO:17; and the VL domain comprises an amino acidsequence that is at least 85%, at least 86%, at least 87%, at least 88%,at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18.In some embodiments, the VH domain comprises an amino acid sequence thatis at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acid sequence of SEQ ID NO:21; and the VLdomain comprises an amino acid sequence that is at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to theamino acid sequence of SEQ ID NO:18. In some embodiments, the VH domaincomprises an amino acid sequence that is at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to the aminoacid sequence of SEQ ID NO:23; and the VL domain comprises an amino acidsequence that is at least 85%, at least 86%, at least 87%, at least 88%,at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18.In some embodiments, the VH domain comprises an amino acid sequence thatis at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acid sequence of SEQ ID NO:13; and the VLdomain comprises an amino acid sequence that is at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to theamino acid sequence of SEQ ID NO:14.

In some embodiments, the VH domain comprises the amino acid sequence ofSEQ ID NO:5; and the VL domain comprises the amino acid sequence of SEQID NO:6. In some embodiments, the VH domain comprises the amino acidsequence of SEQ ID NO:17; and the VL domain comprises the amino acidsequence of SEQ ID NO:18. In some embodiments, the VH domain comprisesthe amino acid sequence of SEQ ID NO:21; and the VL domain comprises theamino acid sequence of SEQ ID NO:18. In some embodiments, the VH domaincomprises the amino acid sequence of SEQ ID NO:23; and the VL domaincomprises the amino acid sequence of SEQ ID NO:18. In some embodiments,the VH domain comprises the amino acid sequence of SEQ ID NO:13; and theVL domain comprises the amino acid sequence of SEQ ID NO:14.

In some embodiments, a binding protein of the present disclosurecomprises an antibody heavy chain comprising the amino acid sequence ofSEQ ID NO:7 and/or an antibody light chain comprising the amino acidsequence of SEQ ID NO:8. In some embodiments, a binding protein of thepresent disclosure comprises an antibody heavy chain comprising theamino acid sequence of SEQ ID NO:19 and/or an antibody light chaincomprising the amino acid sequence of SEQ ID NO:20. In some embodiments,a binding protein of the present disclosure comprises an antibody heavychain comprising the amino acid sequence of SEQ ID NO:22 and/or anantibody light chain comprising the amino acid sequence of SEQ ID NO:20.In some embodiments, a binding protein of the present disclosurecomprises an antibody heavy chain comprising the amino acid sequence ofSEQ ID NO:24 and/or an antibody light chain comprising the amino acidsequence of SEQ ID NO:20. In some embodiments, a binding protein of thepresent disclosure comprises an antibody heavy chain comprising theamino acid sequence of SEQ ID NO:15 and/or an antibody light chaincomprising the amino acid sequence of SEQ ID NO:16.

In some embodiments, a binding protein of the present disclosurecomprises an antibody heavy chain comprising the amino acid sequence ofSEQ ID NO:7 and an antibody light chain comprising the amino acidsequence of SEQ ID NO:8. In some embodiments, a binding protein of thepresent disclosure comprises an antibody heavy chain comprising theamino acid sequence of SEQ ID NO:19 and an antibody light chaincomprising the amino acid sequence of SEQ ID NO:20. In some embodiments,a binding protein of the present disclosure comprises an antibody heavychain comprising the amino acid sequence of SEQ ID NO:22 and an antibodylight chain comprising the amino acid sequence of SEQ ID NO:20. In someembodiments, a binding protein of the present disclosure comprises anantibody heavy chain comprising the amino acid sequence of SEQ ID NO:24and an antibody light chain comprising the amino acid sequence of SEQ IDNO:20. In some embodiments, a binding protein of the present disclosurecomprises an antibody heavy chain comprising the amino acid sequence ofSEQ ID NO:15 and an antibody light chain comprising the amino acidsequence of SEQ ID NO:16.

In some embodiments, the binding proteins comprise an antigen bindingsite comprising: an antibody heavy chain variable (VH) domain comprisinga CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ IDNO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK(SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequenceof ARMFRGAFDY (SEQ ID NO:43); or an antibody light chain variable (VL)domain comprising a CDR-L1 sequence comprising the amino acid sequenceof QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acidsequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising theamino acid sequence of LQDYIYYPT (SEQ ID NO:46). In some embodiments,the binding proteins comprise an antigen binding site comprising: anantibody heavy chain variable (VH) domain comprising a CDR-H1 sequencecomprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42),and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY(SEQ ID NO:43); and an antibody light chain variable (VL) domaincomprising a CDR-L1 sequence comprising the amino acid sequence ofQGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acidsequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising theamino acid sequence of LQDYIYYPT (SEQ ID NO:46).

In some embodiments, the VH domain comprises the sequence, fromN-terminus to C-terminus, FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; whereFR1 comprises the sequence QVQLVESGGGVVQPGRSLRLSCAAS (SEQ ID NO:89),where FR2 comprises the sequence MHWVRQAPGKGLEWVAV (SEQ ID NO:92); whereFR3 comprises the sequence YYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYC (SEQID NO:95); and where FR4 comprises the sequence WGQGTLVTVSS (SEQ IDNO:96). In some embodiments, the VL domain comprises the sequence, fromN-terminus to C-terminus, FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4; whereFR1 comprises the sequence AIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO:98);where FR2 comprises the sequence GWYQQKPGKAPKLLIY (SEQ ID NO:100); whereFR3 comprises the sequence SLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYC (SEQ IDNO:102); and where FR4 comprises the sequence WGQGTLVTVSS (SEQ IDNO:104).

In some embodiments, the VH domain comprises an amino acid sequence thatis at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acid sequence of SEQ ID NO:9; and/or the VLdomain comprises an amino acid sequence that is at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to theamino acid sequence of SEQ ID NO:10.

In some embodiments, the VH domain comprises an amino acid sequence thatis at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acid sequence of SEQ ID NO:9; and the VLdomain comprises an amino acid sequence that is at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to theamino acid sequence of SEQ ID NO:10. In some embodiments, the VH domaincomprises the amino acid sequence of SEQ ID NO:9; and the VL domaincomprises the amino acid sequence of SEQ ID NO:10.

In some embodiments, a binding protein of the present disclosurecomprises an antibody heavy chain comprising the amino acid sequence ofSEQ ID NO:11 or an antibody light chain comprising the amino acidsequence of SEQ ID NO:12. In some embodiments, a binding protein of thepresent disclosure comprises an antibody heavy chain comprising theamino acid sequence of SEQ ID NO:11 and an antibody light chaincomprising the amino acid sequence of SEQ ID NO:12.

In some embodiments, a binding protein of the present disclosurecomprises 1, 2, 3, 4, 5, or 6 CDR sequences of an antibody sequenceshown in Table G. In some embodiments, a binding protein of the presentdisclosure comprises 1, 2, 3, 4, 5, or 6 CDR sequences, a VH domainsequence, and/or a VL domain sequence of an antibody sequence shown inTable H. In some embodiments, a binding protein of the presentdisclosure comprises 1, 2, 3, 4, 5, or 6 CDR sequences, a VH domainsequence, and/or a VL domain sequence of an antibody sequence shown inTable I. In some embodiments, a binding protein of the presentdisclosure comprises 1, 2, 3, or 4 polypeptide sequences shown in TableI.

TABLE G CDR sequences of anti-CD38 binding proteins. Ab CDR_H1 CDR_H2CDR_H3 CDR_L1 CDR_L2 CDR_L3 mAb1 GYTFTSFN IYPGNGGT ARTGGLRRAYFTYESVDSYGNGF LAS QQNKEDPWT (SEQ ID NO: 31) (SEQ ID NO: 32) (SEQ ID NO: 33)(SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36) mAb2 GYTFTSYA IYPGQGGTARTGGLRRAYFTY QSVSSYGQGF GAS QQNKEDPWT (SEQ ID NO: 37) (SEQ ID NO: 38)(SEQ ID NO: 33) (SEQ ID NO: 39) (SEQ ID NO: 40) (SEQ ID NO: 36) mAb3GYTFTSFN IYPGNGGT ARTGGLRRAYFTY ESVDSYGNGF LAS QQNKEDPWT (SEQ ID NO: 31)(SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 35)(SEQ ID NO: 36) mAb4 GYTFTSFN IYPGNGGT ARTGGLRRAYFTY ESVDSYGNGF LASQQNKEDPWT (SEQ ID NO: 31) (SEQ ID NO: 32) (SEQ ID NO: 33)(SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36) mAb5 GYTFTSFN IYPGNGGTARTGGLRRAYFTY ESVDSYGNGF LAS QQNKEDPWT (SEQ ID NO: 31) (SEQ ID NO: 32)(SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36) mAb6GFTFSSYG IWYDGSNK ARMFRGAFDY QGIRND AAS LQDYIYYPT (SEQ ID NO: 41)(SEQ ID NO: 42) (SEQ ID NO: 43) (SEQ ID NO: 44) (SEQ ID NO: 45)(SEQ ID NO: 46)

TABLE HVariable domain sequences of anti-CD38 (mAb1-7) and other binding proteins.Ab VH (protein) VL (protein) mAb1 QVQLQQSGAELVRSGASVKMSCKADIVLTQSPASLAVSLGQRATISCR S GYTFTSFN MHWVKETPGQGLEWI AS ESVDSYGNGFMHWYQQKPG GY IYPGNGGT NYNQKFKGKATLT QPPKLLIY LAS NLESGVPARFSGADTSSSTAYMQISSLTSEDSAVYFC SGSRTDFTLTIDPVEADDAATYY ARTGGLRRAYFTYWGQGTLVTVS C QQ NKEDPWT FGGGTKLEIK S (SEQ ID NO: 5) (SEQ ID NO: 6 mAb2QVQLVQSGAEVVKPGASVKVSCKA DIVLTQSPATLSLSPGERATISCR S GYTFTSVAMHWVKEAPGQRLEW AS QSVSSYGQGF MHWYQQKPG IGY IYPGQGGT NYNQKFQGRATLTQPPRLLIY GAS SRAT ADTSASTAYMELSSLRSEDTAVYFC GIPARFSGSGSGTDFTLTISPLEPEARTGGLRRAYFTY WGQGTLVTVS DFAVYYC QQNKEDPWT FGGGT S (SEQ ID NO: 13)KLEIK (SEQ ID NO: 14) mAb3 QVQLVQSGAEVVKPGASVKVSCKADIVLTQSPATLSLSPGERATISCR S GYTFTSFN MHWVKEAPGQRLEWI AS ESVDSYGNGFMHWYQQKPG GY IYPGNGGT NYNQKFQGRATLTA QPPRLLIY LAS SRATDTSASTAYMELSSLRSEDTAVYFC A GIPARFSGSGSGTDFTLTISPLEPE RTGGLRRAYFTYWGQGTLVTVSS DFAVYYC QQNKEDPWT FGGGT (SEQ ID NO: 17)KLEIK (SEQ ID NO: 18) mAb4 QVQLVQSGAEVVKSGASVKVSCKADIVLTQSPATLSLSPGERATISCR S GYTFTSFN MHWVKEAPGQGLEWI AS ESVDSYGNGFMHWYQQKPG GY IYPGNGGT NYNQKFQGRATLTA QPPRLLIY LAS SRATDTSASTAYMEISSLRSEDTAVYFC A GIPARFSGSGSGTDFTLTISPLEPE RTGGLRRAYFTYWGQGTLVTVSS DFAVYYC QQNKEDPWT FGGGT (SEQ ID NO: 21)KLEIK (SEQ ID NO: 18) mAb5 QVQLVQSGAEVVKPGASVKMSCKDIVLTQSPATLSLSPGERATISCR AS GYTFTSFN MHWVKEAPGQRLE AS ESVDSYGNGFMHWYQQKPG WIGY IYPGNGGT NYNQKFQGRATL QPPRLLIY LAS SRATTADTSASTAYMEISSLRSEDTAVYF GIPARFSGSGSGTDFTLTISPLEPE C ARTGGLRRAYFTYWGQGTLVTV DFAVYYC QQ NKEDPWT FGGGT SS (SEQ ID NO: 23)KLEIK (SEQ ID NO: 18) mAb6 QVQLVESGGGVVQPGRSLRLSCAAAIQMTQSPSSLSASVGDRVTITC S GFTFSSYG MHWVRQAPGKGLEW RAS Q GIRNDLGWYQQKPGKAP VAV IWYDGSNK YYADSVKGRFTIS KLLIY AAS SLQSGVPSRFSGSGSGDNSKNTLYLQMNSLRAEDTAVYY GTDFTLTISGLQPEDSATYY LQ C ARMFRGAFDYWGQGTLVTVSS DYIYYPT FGQGTKVEIK(SEQID (SEQ ID NO: 9) NO: 10) mAb7QVQLVQSGAEVAKPGTSVKLSCKA DIVMTQSHLSMSTSLGDPVSITC SGYTFTDYWMQWVKQRPGQGLEWKASQDVSTVVAWYQQKPGQSP IGTIYPGDGDTGYAQKFQGKATLTA RRLIYSASYRYIGVPDRFTGSGADKSSKTVYMHLSSLASEDSAVYYC GTDFTFTISSVQAEDLAVYYCQQARGDYYGSNSLDYWGQGTSVTVSS HYSPPYTFGGGTKLEIK (SEQ ID (SEQ ID NO: 47)NO: 48) Anti- QVQLVQSGAEVVKPGASVKVSCKA DIQMTQSPSSLSASVGDRVTITCCD28_(sup) S GYTFTSYY IHWVRQAPGQGLEWI QAS Q NIYVW LNWYQQKPGKAP GSIYPGNVNT NYAQKFQGRATLTV KLLIY KAS NLHTGVPSRFSGSGSDTSISTAYMELSRLRSDDTAVYYC T GTDFTLTISSLQPEDIATYYC QQ RSHYGLDWNFDVWGKGTTVTVSS GQTYPYT FGQGTKLEIK (SEQ (SEQ ID NO: 49) ID NO: 50) Anti-QVQLQESGPGLVKPSQTLSLTCTVS DIVLTQSPASLAVSPGQRATITCR CD28_(evn) GFSLSDYGVHWVRQPPGKGLEWL AS ESVEYYVTSL MQWYQQKPG GV IWAGGGT NYNPSLKSRKTISKDQPPKLLIF AAS NVESGVPARFSG TSKNQVSLKLSSVTAADTAVYYC ASGSGTDFTLTINPVEANDVANYY RDKGYSYYYSMDY WGQGTTVTVS C QQ SRKVPYT FGQGTKLEIKS (SEQ ID NO: 51) (SEQ ID NO: 52) Anti- QVQLVESGGGVVQPGRSLRLSCAADIVMTQTPLSLSVTPGQPASISCK CD3_(mid) S GFTFTKAW MHWVRQAPGKQLE SSQSLVHNNANTY LSWYLQKP WVAQ IKDKSNSYAT YYADSVKGR GQSPQSLIY KVS NRFSGVPDRFSFTISRDDSKNTLYLQMNSLRAEDTA GSGSGTDFTLKISRVEAEDVGVY VYYC RGVYYALSPFDYWGQGTLV YC GQGTQYPFT FGSGTKVEIK TVSS (SEQ ID NO: 53) (SEQ ID NO: 54)Anti- QVQLVESGGGVVQPGRSLRLSCAA DIVMTQTPLSLSVTPGQPASISCK CD3_(low) SGFTFTKAW MHWVRQAPGKGLE SS QSLVHNNGNTY LSWYLQKP WVAQI KDKSNSYAT YYADSVKGRGQSPQLLIY KVS NRFSGVPDRFS FTISRDNSKNTLYLQMNSLRAEDTAGSGSGTDFTLKISRVEAEDVGVY VYYCR GVYYALSPFDY WGQGTLV YC GQGTQYPFTFGGGTKVEIK TVSS (SEQ ID NO: 84) (SEQ ID NO: 85) Note: CDR sequences arebolded and underlined in amino acid sequences above.

TABLE I Full-length sequences of binding proteins.mAb2xCD28supxCD3mid IgG4 FALA CD28supxCD3midQVQLVQSGAEVVKPGASVKVSCKASGYTF SEQ ID IgG4(hole) FALA HeavyTSYYIHWVRQAPGQGLEWIGSIYPGNVNTN NO: 60 Chain 1YAQKFQGRATLTVDTSISTAYMELSRLRSD (e.g., a second polypeptideDTAVYYCTRSHYGLDWNFDVWGKGTTVT chain of a trispecific bindingVSSSQVQLVESGGGVVQPGRSLRLSCAASG protein of the presentFTFTKAWMHWVRQAPGKQLEWVAQIKDK disclosure) SNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWG QGTLVTVSSRTASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCT LPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGCD28supxCD3mid Light DIVMTQTPLSLSVTPGQPASISCKSSQSLVH SEQ ID Chain 1NNANTYLSWYLQKPGQSPQSLIYKVSNRFS NO: 61 (e.g., a first polypeptideGVPDRFSGSGSGTDFTLKISRVEAEDVGVY chain of a trispecific bindingYCGQGTQYPFTFGSGTKVEIKGQPKAAPDI protein of the presentQMTQSPSSLSASVGDRVTITCQASQNIYVW disclosure)LNWYQQKPGKAPKLLIYKASNLHTGVPSRF SGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGECmAb2 IgG4(knob) FALA QVQLVQSGAEVVKPGASVKVSCKASGYTF SEQ ID Heavy Chain 2TSYAMHWVKEAPGQRLEWIGYIYPGQGGT NO: 62 (e.g., a third polypeptideNYNQKFQGRATLTADTSASTAYMELSSLRS chain of a trispecific bindingEDTAVYFCARTGGLRRAYFTYWGQGTLVT protein of the presentVSSASTKGPSVFPLAPCSRSTSESTAALGCL disclosure)VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEM TKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGmAb2 Light Chain 2 DIVLTQSPATLSLSPGERATISCRASQSVSSY SEQ ID(e.g., a fourth polypeptide GQGFMHWYQQKPGQPPRLLIYGASSRATGI NO: 63chain of a trispecific binding PARFSGSGSGTDFTLTISPLEPEDFAVYYCQprotein of the present QNKEDPWTFGGGTKLEIKRTVAAPSVFIFPP disclosure)SDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC mAb2xCD28supxCD3mid IgG1LALA P329ACD28supxCD3mid QVQLVQSGAEVVKPGASVKVSCKASGYTF SEQ IDIgG1(hole) LALA P329A TSYYIHWVRQAPGQGLEWIGSIYPGNVNTN NO: 64Heavy Chain 1 YAQKFQGRATLTVDTSISTAYMELSRLRSD (e.g., a second polypeptideDTAVYYCTRSHYGLDWNFDVWGKGTTVT chain of a trispecific bindingVSSSQVQLVESGGGVVQPGRSLRLSCAASG protein of the presentFTFTKAWMHWVRQAPGKQLEWVAQIKDK disclosure) SNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWG QGTLVTVSSRTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG CD28supxCD3mid Light See above. SEQ ID Chain 1 NO: 61(e.g., a first polypeptide chain of a trispecific bindingprotein of the present disclosure) mAb2 IgG1(knob) LALAQVQLVQSGAEVVKPGASVKVSCKASGYTF SEQ ID P329A Heavy Chain 2TSYAMHWVKEAPGQRLEWIGYIYPGQGGT NO: 65 (e.g., a third polypeptideNYNQKFQGRATLTADTSASTAYMELSSLRS chain of a trispecific bindingEDTAVYFCARTGGLRRAYFTYWGQGTLVT protein of the presentVSSASTKGPSVFPLAPSSKSTSGGTAALGCL disclosure)VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPCRD ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLS PGmAb2 Light Chain 2 See above. SEQ ID (e.g., a fourth polypeptide NO: 63chain of a trispecific binding protein of the present disclosure)mAb2xCD28supxCD3mid IgG1 NNSA CD28supxCD3midQVQLVQSGAEVVKPGASVKVSCKASGYTF SEQ ID IgG1(hole) NNSA HeavyTSYYIHWVRQAPGQGLEWIGSIYPGNVNTN NO: 66 Chain 1YAQKFQGRATLTVDTSISTAYMELSRLRSD (e.g., a second polypeptideDTAVYYCTRSHYGLDWNFDVWGKGTTVT chain of a trispecific bindingVSSSQVQLVESGGGVVQPGRSLRLSCAASG protein of the presentFTFTKAWMHWVRQAPGKQLEWVAQIKDK disclosure) SNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWG QGTLVTVSSRTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHICPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGCD28supxCD3mid Light See above. SEQ ID Chain 1 NO: 61(e.g., a first polypeptide chain of a trispecific bindingprotein of the present disclosure) mAb2 IgG1(knob) NNSAQVQLVQSGAEVVKPGASVKVSCKASGYTF SEQ ID Heavy Chain 2TSYAMHWVKEAPGQRLEWIGYIYPGQGGT NO: 67 (eg., a third polypeptideNYNQKFQGRATLTADTSASTAYMELSSLRS chain of a trispecific bindingEDTAVYFCARTGGLRRAYFTYWGQGTLVT protein of the presentVSSASTKGPSVFPLAPSSKSTSGGTAALGCL disclosure)VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGCD38VH1 Light Chain 2 See above. SEQ ID (e.g., a fourth polypeptideNO: 63 chain of a trispecific binding protein of the present disclosure)mAb6xCD28supxCD3mid IgG4 FALA CD28supxCD3mid See above. SEQ IDIgG4(hole) FALA Heavy NO: 60 Chain 1 (e.g., a second polypeptidechain of a trispecific binding protein of the present disclosure)CD28supxCD3mid Light See above. SEQ ID Chain 1 NO: 61(e.g., a first polypeptide chain of a trispecific bindingprotein of the present disclosure) mAb6 IgG4(knob) FALAQVQLVESGGGVVQPGRSLRLSCAASGFTFS SEQ ID Heavy Chain 2SYGMHWVRQAPGKGLEWVAVIWYDGSNK NO: 68 (e.g., a third polypeptideYYADSVKGRFTISGDNSKNTLYLQMNSLRA chain of a trispecific bindingEDTAVYYCARMFRGAFDYWGQGTLVTVSS protein of the presentASTKGPSVFPLAPCSRSTSESTAALGCLVKD disclosure)YFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQ VSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG mAb6 Light2AIQMTQSPSSLSASVGDRVTITCRASQGIRN SEQ ID (e.g., a fourth polypepfideDLGWYQQKPGKAPKLLIYAASSLQSGVPSR NO: 69 chain of a trispecific bindingFSGSGSGTDFTLTISGLQPEDSATYYCLQDY protein of the presentIYYPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ disclosure)LKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C mAb6xCD28supxCD3mid IgG1LALA P329ACD28supxCD3mid See above. SEQ ID IgG1(hole) LALA P329A NO: 64Heavy Chain 1 (e.g., a second polypeptide chain of a trispecific bindingprotein of the present disclosure) CD28supxCD3mid Light See above.SEQ ID Chain 1 NO: 61 (e.g., a first polypeptidechain of a trispecific binding protein of the present disclosure)mAb6 IgG1(knob) LALA QVQLVESGGGVVQPGRSLRLSCAASGFTFS SEQ IDP329A Heavy Chain 2 SYGMHWVRQAPGKGLEWVAVIWYDGSNK NO: 70(e.g., a third polypeptide YYADSVKGRFTISGDNSKNTLYLQMNSLRAchain of a trispecific binding EDTAVYYCARMFRGAFDYWGQGTLVTVSSprotein of the present ASTKGPSVFPLAPSSKSTSGGTAALGCLVK disclosure)DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPCRDE LTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP GmAb6 Light2 See above. SEQ ID (e.g., a fourth polypeptide NO: 69chain of a trispecific binding protein of the present disclosure)mAb6xCD28supxCD3mid IgG1 NNSA CD28supxCD3mid See above. SEQ IDIgG1(hole) Heavy NO: 66 Chain 1 (e.g., a second polypeptidechain of a trispecific binding protein of the present disclosure)CD28supxCD3mid Light See above. SEQ ID Chain 1 NO: 61(e.g., a first polypeptide chain of a trispecific bindingprotein of the present disclosure) mAb6 IgG1(knob) NNSAQVQLVESGGGVVQPGRSLRLSCAASGFTFS SEQ ID Heavy Chain 2SYGMHWVRQAPGKGLEWVAVRVYDGSNK NO: 71 (e.g., a third polypeptideYYADSVKGRFTISGDNSKNTLYLQMNSLRA chain of a trispecific bindingEDTAVYYCARMFRGAFDYWGQGTLVTVSS protein of the presentASTKGPSVFPLAPSSKSTSGGTAALGCLVK disclosure)DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN NASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRD ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLS PGmAb6 Light2 See above. SEQ ID (e.g., a fourth polypeptide NO: 69chain of a trispecific binding protein of the present disclosure)mAb1 monovalent antibody mAb1 heavy chain QVQLQQSGAELVRSGASVKMSCKASGYTFSEQ ID TSFNMHWVKETPGQGLEWIGYIYPGNGGT NO: 7NYNQKFKGKATLTADTSSSTAYMQISSLTS EDSAVYFCARTGGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G mAb1 light chainDIVLTQSPASLAVSLGQRATISCRASESVDS SEQ ID YGNGFMHWYQQKPGQPPKLLIYLASNLESNO: 8 GVPARFSGSGSRTDFTLTIDPVEADDAATY YCQQNKEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC mAb2 monovalent antibodymAb2 heavy chain QVQLVQSGAEVVKPGASVKVSCKASGYTF SEQ IDTSYAMHWVKEAPGQRLEWIGYIYPGQGGT NO: 15 NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLA GPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP GmAb2 light chain DIVLTQSPATLSLSPGERATISCRASQSVSSY SEQ IDGQGFMHWYQQKPGQPPRLLIYGASSRAT NO: 16 GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECmAb3 monovalent antibody mAb3 heavy chain QVQLVQSGAEVVKPGASVKVSCKASGYTFSEQ ID TSFNMHWVKEAPGQRLEWIGYIYPGNGGT NO: 19NYNQKFQGRATLTADTSASTAYMELSSLRS EDTAVYFCARTGGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G mAb3 light chainDIVLTQSPATLSLSPGERATISCRASESVDSY SEQ ID GNGFMHWYQQKPGQPPRLLIYLASSRATNO: 20 GIPARFSGSGSGTDFTLTISPLEPEDFAVYYC QQNKEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC mAb4 monovalent antibodymAb4 heavy chain QVQLVQSGAEVVKSGASVKVSCKASGYTF SEQ IDTSFNMHWVKEAPGQGLEWIGYIYPGNGGT NO: 22 NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLA GPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP GmAb4 light chain DIVLTQSPATLSLSPGERATISCRASESVDSY SEQ IDGNGFMHWYQQKPGQPPRLLIYLASSRAT NO: 20 GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECmAb5 monovalent antibody mAb5 heavy chain QVQLVQSGAEVVKPGASVKMSCKASGYTFSEQ ID TSFNMHWVKEAPGQRLEWIGYIYPGNGGT NO: 24NYNQKFQGRATLTADTSASTAYMEISSLRS EDTAVYFCARTGGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G mAb5 light chainDIVLTQSPATLSLSPGERATISCRASESVDSY SEQ ID GNGFMHWYQQKPGQPPRLLIYLASSRATNO: 20 GIPARFSGSGSGTDFTLTISPLEPEDFAVYYC QQNKEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC mAb6 monovalent antibodymAb6 heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFS SEQ IDSYGMHWVRQAPGKGLEWVAVIWYDGSNK NO: 11 YYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGP DVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVICFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGmAb6 light chain AIQMTQSPSSLSASVGDRVTITCRASQGIRN SEQ IDDLGWYQQKPGKAPKLLIYAASSLQSGVPS NO: 12 RFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGECmAb7 monovalent antibody mAb7 heavy chain QVQLVQSGAEVAKPGTSVKLSCKASGYTFTSEQ ID DYWMQWVKQRPGQGLEWIGTIYPGDGDT NO: 107GYAQKFQGKATLTADKSSKTVYMHLSSLA SEDSAVYYCARGDYYGSNSLDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK mAb7 light chainDIVMTQSHLSMSTSLGDPVSITCKASQDVST SEQ ID VVAWYQQKPGQSPRRLIYSASYRYIGVPDRNO: 106 FTGSGAGTDFTFTISSVQAEDLAVYYCQQH YSPPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

TABLE J Full-length polynucleotide sequences of binding proteins.mAb2xCD28supxCD3mid IgG4 FALA CD28supxCD3midCAGGTGCAGCTGGTGCAGTCTGGCGCCGA SEQ ID IgG4(hole) FALA HeavyGGTCGTGAAACCTGGCGCCTCTGTGAAGG NO: 72 Chain 1TGTCCTGCAAGGCCAGCGGCTACACCTTT (e.g., encoding a secondACCAGCTACTACATCCACTGGGTGCGCCA polypeptide chain of aGGCCCCTGGACAGGGACTGGAATGGATC trispecific binding protein ofGGCAGCATCTACCCCGGCAACGTGAACA the present disclosure)CCAACTACGCCCAGAAGTTCCAGGGCAG AGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAACTGAGCCGGCTG AGAAGCGACGACACCGCCGTGTACTACTGCACCCGGTCCCACTACGGCCTGGATTGG AACTTCGACGTGTGGGGCAAGGGCACCACCGTGACAGTGTCTAGCAGCCAGGTGCAG CTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCAGAAGCCTGAGACTGAGCTG TGCCGCCAGCGGCTTCACCTTCACCAAGGCCTGGATGCACTGGGTGCGCCAGGCCCCT GGAAAGCAGCTGGAATGGGTGGCCCAGATCAAGGACAAGAGCAACAGCTACGCCAC CTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGA ACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTG TCGGGGCGTGTACTATGCCCTGAGCCCCTTCGATTACTGGGGCCAGGGAACCCTCGTG ACCGTGTCTAGTCGGACCGCCAGCACAAAGGGCCCATCGGTGTTCCCTCTGGCCCCTT GCAGCAGAAGCACCAGCGAATCTACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACT TTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACAC CTTTCCAGCCGTGCTCCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTG CCCAGCAGCAGCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCAG CAACACCAAGGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTCCTTG CCCAGCCCCTGAAGCTGCCGGCGGACCCTCCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCC AGGAAGATCCCGAGGIGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAAC GCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTG CTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCC AACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCC CCGCGAGCCTCAAGTGTGTACCCTGCCCCCTAGCCAGGAAGAGATGACCAAGAACCA GGTGTCCCTGAGCTGTGCCGTGAAAGGCTTCTACCCCAGCGACATTGCCGTGGAATGG GAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGC GACGGCTCATTCTTCCTGGTGTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGGAA GGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG AAGTCCCTGTCTCTGTCCCTGGGCCD28supxCD3mid Light GACATCGTGATGACCCAGACCCCCCTGAG SEQ ID Chain 1CCTGAGCGTGACACCTGGACAGCCTGCCA NO: 73 (e.g., encoding a firstGCATCAGCTGCAAGAGCAGCCAGAGCCT polypeptide chain of aGGTGCACAACAACGCCAACACCTACCTG trispecific binding protein ofAGCTGGTATCTGCAGAAGCCCGGCCAGA the present disclosure)GCCCCCAGTCCCTGATCTACAAGGTGTCC AACAGATTCAGCGGCGTGCCCGACAGATTCTCCGGCAGCGGCTCTGGCACCGACTTCA CCCTGAAGATCAGCCGGGTGGAAGCCGAGGACGTGGGCGTGTACTATTGTGGCCAGG GCACCCAGTACCCCTTCACCTTTGGCAGCGGCACCAAGGTGGAAATCAAGGGCCAGC CCAAGGCCGCCCCCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGT GGGCGACAGAGTGACCATCACCTGTCAGGCCAGCCAGAACATCTACGTGTGGCTGAA CTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAA CCTGCACACCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACC CTGACAATCAGCTCCCTGCAGCCCGAGGACATTGCCACCTACTACTGCCAGCAGGGCC AGACCTACCCCTACACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGACCAAGGGCC CCAGCCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCACCTAGCGACGAGCAGCT GAAGTCCGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCC AAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGT GACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACACTGAG CAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCT GTCTAGCCCCGTGACCAAGAGCTTCAACC GGGGCGAGTGTmAb2 IgG4(knob) FALA CAGGTGCAGCTGGTGCAGTCTGGCGCCGA SEQ ID Heavy Chain 2AGTCGTGAAACCTGGCGCCTCCGTGAAGG NO: 74 (e.g., encoding a thirdTGTCCTGCAAGGCCAGCGGCTACACCTTT polypeptide chain of aACCAGCTACGCCATGCACTGGGTCAAAG trispecific binding protein ofAGGCCCCTGGCCAGAGACTGGAATGGAT the present disclosure)CGGCTACATCTACCCCGGCCAGGGCGGCA CCAACTACAACCAGAAGTTCCAGGGCAGAGCCACCCTGACCGCCGATACAAGCGCC AGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGATACCGCCGTGTACTTCT GTGCCAGAACAGGCGGCCTGAGGCGGGCCTACTTTACCTATTGGGGCCAGGGCACCC TCGTGACCGTGTCTAGCGCTAGCACAAAGGGCCCATCGGTGTTCCCTCTGGCCCCTTG CAGCAGAAGCACCAGCGAATCTACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTT TCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACC TTTCCAGCCGTGCTCCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGC CCAGCAGCAGCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCAGC AACACCAAGGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTCCTTGC CCAGCCCCTGAAGCTGCCGGCGGACCCTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGG ACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCA GGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACACGC CAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCT GACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCA ACAAGGGCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCC CGCGAGCCTCAAGTGTATACCCTGCCCCCTTGCCAGGAAGAGATGACCAAGAACCAG GTGTCCCTGTGGTGTCTCGTGAAAGGCTTCTACCCCAGCGACATTGCCGTGGAATGGG AGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCG ACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGGAAG GCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGA AGTCCCTGTCTCTGTCCCTGGGC ntAb2 Light Chain 2GACATCGTGCTGACACAGAGCCCTGCCAC SEQ ID (e.g., encoding a fourthCCTGTCTCTGAGCCCTGGCGAGAGAGCCA NO: 75 polypeptide chain of aCCATCAGCTGTAGAGCCAGCCAGAGCGT trispecific binding protein ofGTCCAGCTACGGCCAGGGCTTCATGCACT the present disclosure)GGTATCAGCAGAAGCCCGGCCAGCCCCC CAGACTGCTGATCTATGGCGCCAGCAGCAGAGCCACAGGCATCCCCGCCAGATTTTCT GGCTCTGGCAGCGGCACCGACTTCACCCTGACAATCAGCCCCCTGGAACCCGAGGAC TTCGCCGTGTACTACTGCCAGCAGAACAAAGAGGACCCCTGGACCTTCGGCGGAGGC ACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCACCT AGCGACGAGCAGCTGAAGTCCGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTC TACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAA CAGCCAGGAAAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCA GCACCCTGACCCTGTCCAAGGCCGATTACGAGAAGCACAAGGTGTACGCCTGCGAAG TGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC mAb2xCD28supxCD3mid IgG1LALA P329ACD28supxCD3mid CAGGTGCAGCTGGTGCAGTCTGGCGCCGA SEQ IDIgG1(hole) LALA P329A GGTCGTGAAACCTGGCGCCTCTGTGAAGG NO: 76 Heavy Chain 1TGTCCTGCAAGGCCAGCGGCTACACCTTT (e.g., encoding a secondACCAGCTACTACATCCACTGGGTGCGCCA polypeptide chain of aGGCCCCTGGACAGGGACTGGAATGGATC trispecific binding protein ofGGCAGCATCTACCCCGGCAACGTGAACA the present disclosure)CCAACTACGCCCAGAAGTTCCAGGGCAG AGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAACTGAGCCGGCTG AGAAGCGACGACACCGCCGTGTACTACTGCACCCGGTCCCACTACGGCCTGGATTGG AACTTCGACGTGTGGGGCAAGGGCACCACCGTGACAGTGTCTAGCAGCCAGGTGCAG CTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCAGAAGCCTGAGACTGAGCTG TGCCGCCAGCGGCTTCACCTTCACCAAGGCCTGGATGCACTGGGTGCGCCAGGCCCCT GGAAAGCAGCTGGAATGGGTGGCCCAGATCAAGGACAAGAGCAACAGCTACGCCAC CTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGA ACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTG TCGGGGCGTGTACTATGCCCTGAGCCCCTTCGATTACTGGGGCCAGGGAACCCTCGTG ACCGTGTCTAGTCGGACCGCCAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTA GCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACT TTCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACCAGCGGCGTGCACAC CTTTCCAGCTGTGCTGCAGTCCAGCGGCCTGTACAGCCTGAGCAGCGTCGTGACAGTG CCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGC AACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTG TCCCCCTTGTCCTGCCCCCGAAGCCGCCGGAGGCCCTTCCGTGTTCCTGTTCCCCCCA AAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTG GATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAA GTGCACAACGCCAAGACCAAGCCAAGAGAGGAACAGTACAACAGCACCTACCGGGT GGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTG CAAGGTGTCCAACAAGGCCCTGGCCGCCCCCATCGAGAAAACCATCAGCAAGGCCAA GGGCCAGCCCCGCGAACCCCAGGTGTGCACACTGCCCCCAAGCAGGGACGAGCTGA CCAAGAACCAGGTGTCCCTGAGCTGTGCCGTGAAAGGCTTCTACCCCTCCGATATCGC CGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACAGCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCCGG TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACT ACACCCAGAAGTCCCTGAGCCTGAGCCCC GGCCD28supxCD3mid Light See above. SEQ ID Chain 1 NO: 73(e.g., encoding a first polypeptide chain of atrispecific binding protein of the present disclosure)mAb2 IgG1(knob) LALA CAGGTGCAGCTGGTGCAGTCTGGCGCCGA SEQ IDP329A Heavy Chain 2 AGTCGTGAAACCTGGCGCCTCCGTGAAGG NO: 77(e.g., encoding a third TGTCCTGCAAGGCCAGCGGCTACACCTTTpolypeptide chain of a ACCAGCTACGCCATGCACTGGGTCAAAGtrispecific binding protein of AGGCCCCTGGCCAGAGACTGGAATGGATthe present disclosure) CGGCTACATCTACCCCGGCCAGGGCGGCACCAACTACAACCAGAAGTTCCAGGGCAG AGCCACCCTGACCGCCGATACAAGCGCCAGCACCGCCTACATGGAACTGAGCAGCCT GCGGAGCGAGGATACCGCCGTGTACTTCTGTGCCAGAACAGGCGGCCTGAGGCGGGC CTACTTTACCTATTGGGGCCAGGGCACCCTCGTGACCGTGTCTAGCGCTAGCACAAAG GGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCC GCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAATT CTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCAGCGGCCT GTACAGCCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTAC ATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACC CAAGAGCTGCGACAAGACCCACACCTGTCCCCCTrGTCCTGCCCCCGAAGCCGCCGG AGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCG GACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAA GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCAAGAGA GGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGA CTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGCCGCCCC CATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACA CACTGCCCCCATGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGT GAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGA GAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTAC TCCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCTCCG TGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGG C mAb2 Light Chain 2 See above. SEQ ID(e.g., encoding a fourth NO: 75 polypeptide chain of atrispecific binding protein of the present disclosure)mAb2xCD28supxCD3mid IgG1 NNSA CD28supxCD3midCAGGTGCAGCTGGTGCAGTCTGGCGCCGA SEQ ID IgG1(hole) NNSA HeavyGGTCGTGAAACCTGGCGCCTCTGTGAAGG NO: 78 Chain 1TGTCCTGCAAGGCCAGCGGCTACACCTTT (e.g., encoding a secondACCAGCTACTACATCCACTGGGTGCGCCA polypeptide chain of aGGCCCCTGGACAGGGACTGGAATGGATC trispecific binding protein ofGGCAGCATCTACCCCGGCAACGTGAACA the present disclosure)CCAACTACGCCCAGAAGTTCCAGGGCAG AGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAACTGAGCCGGCTG AGAAGCGACGACACCGCCGTGTACTACTGCACCCGGTCCCACTACGGCCTGGATTGG AACTTCGACGTGTGGGGCAAGGGCACCACCGTGACAGTGTCTAGCAGCCAGGTGCAG CTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCAGAAGCCTGAGACTGAGCTG TGCCGCCAGCGGCTTCACCTTCACCAAGGCCTGGATGCACTGGGTGCGCCAGGCCCCT GGAAAGCAGCTGGAATGGGTGGCCCAGATCAAGGACAAGAGCAACAGCTACGCCAC CTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGA ACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTG TCGGGGCGTGTACTATGCCCTGAGCCCCTTCGATTACTGGGGCCAGGGAACCCTCGTG ACCGTGTCTAGTCGGACCGCCAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTA GCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACT TTCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACCAGCGGCGTGCACAC CTTTCCAGCTGTGCTGCAGTCCAGCGGCCTGTACAGCCTGAGCAGCGTCGTGACAGTG CCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGC AACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTG TCCCCCTTGTCCTGCCCCCGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCA AAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTG GATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAA GTGCACAACGCCAAGACCAAGCCAAGAGAGGAACAGTACAACAATGCCTCCCGGGT GGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTG CAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAA GGGCCAGCCCCGCGAACCCCAGGTGTGCACACTGCCCCCAAGCAGGGACGAGCTGA CCAAGAACCAGGTGTCCCTGAGCTGTGCCGTGAAAGGCTTCTACCCCTCCGATATCGC CGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACAGCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCCGG TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACT ACACCCAGAAGTCCCTGAGCCTGAGCCCC GGCCD28supxCD3mid Light See above. SEQ ID Chain 1 NO: 73(e.g., encoding a first polypeptide chain of atrispecific binding protein of the present disclosure)mAb2 IgG1(knob) NNSA CAGGTGCAGCTGGTGCAGTCTGGCGCCGA SEQ ID Heavy Chain 2AGTCGTGAAACCTGGCGCCTCCGTGAAGG NO: 79 (e.g., encoding a thirdTGTCCTGCAAGGCCAGCGGCTACACCTTT polypeptide chain of aACCAGCTACGCCATGCACTGGGTCAAAG trispecific binding protein ofAGGCCCCTGGCCAGAGACTGGAATGGAT the present disclosure)CGGCTACATCTACCCCGGCCAGGGCGGCA CCAACTACAACCAGAAGTTCCAGGGCAGAGCCACCCTGACCGCCGATACAAGCGCC AGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGATACCGCCGTGTACTTCT GTGCCAGAACAGGCGGCCTGAGGCGGGCCTACTTTACCTATTGGGGCCAGGGCACCC TCGTGACCGTGTCTAGCGCTAGCACAAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTT CCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCA ACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCC CACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTATGTTGACGGCGTGGAGGTG CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAATGCCTCCCGTGTGGTC AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAG AATCAAGTCAGCCTGTGGTGCCTGGTAAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG ACTCCGACGGCTCCTTCTTCCTCTACTCAAAACTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG CAGAAGAGCCTCTCCCTGTCTCCGGGTmAb2 Light Chain 2 See above. SEQ ID (e.g., encoding a fourth NO: 75polypeptide chain of a trispecific binding protein ofthe present disclosure) mAb6xCD28supxCD3mid IgG4 FALA CD28supxCD3midSee above. SEQ ID IgG4(hole) FALA Heavy NO: 72 Chain 1(e.g., encoding a second polypeptide chain of atrispecific binding protein of the present disclosure)CD28supxCD3mid Light See above. SEQ ID Chain 1 NO: 73(e.g., encoding a first polypeptide chain of atrispecific binding protein of the present disclosure)mAb6 IgG4(knob) FALA CAGGTGCAGCTGGTGGAAAGCGGCGGAG SEQ ID Heavy Chain 2GCGTGGTGCAGCCTGGCAGGTCTCTGAGA NO: 80 (e.g., encoding a thirdCTGAGCTGTGCCGCCAGCGGCTTCACCTT polypeptide chain of aCAGCAGCTACGGAATGCACTGGGTGCGC trispecific binding protein ofCAGGCCCCTGGCAAAGGACTGGAATGGC the present disclosure)TGGCCGTGATTTGGTACGACGGCAGCAAC AAGTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGGCGACAACAGCAA GAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTAC TGCGCCAGAATGTTCAGAGGCGCCTTCGACTACTGGGGCCAGGGCACACTCGTGACCG TGTCTAGTGCGTCGACCAAGGGCCCATCGGTGTTCCCTCTGGCCCCTTGCAGCAGAAG CACCAGCGAATCTACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCC GTGACCGTGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCCG TGCTCCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCA GCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGG TGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTCCTTGCCCAGCCCCTG AAGCTGCCGGCGGACCCTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGAT GATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCC CGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAA GCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCT GCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCC TGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCTC AAGTGTATACCCTGCCCCCTTGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGT GGTGTCTCGTGAAAGGCTTCTACCCCAGCGACATTGCCGTGGAATGGGAGAGCAACG GCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCAT TCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGGAAGGCAACGTGT TCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTC TCTGTCCCTGGGC mAb6 Light2GCCATCCAGATGACCCAGAGCCCCAGCA SEQ ID (e.g., encoding a fourthGCCTGTCTGCCAGCGTGGGCGACAGAGTG NO: 81 polypeptide chain of aACCATCACCTGTAGAGCCAGCCAGGGCAT trispecific binding protein ofCCGGAACGACCTGGGCTGGTATCAGCAG the present disclosure)AAGCCTGGCAAGGCCCCCAAGCTGCTGAT CTACGCCGCTAGCTCTCTGCAGTCCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCC GGCACCGACTTCACCCTGACAATCTCTGGCCTGCAGCCCGAGGACAGCGCCACCTACT ACTGTCTGCAAGACTACATCTACTACCCCACCTTCGGCCAGGGCACCAAGGTGGAAA TCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCACCTAGCGACGAGCAGCT GAAGTCCGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCC AAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGT GACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACACTGAG CAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCT GTCTAGCCCCGTGACCAAGAGCTTCAACC GGGGCGAGTGTmAb6xCD28supxCD3mid IgG1LALA P329A CD28supxCD3mid See above. SEQ IDIgG1(hole) LALA P329A NO: 76 Heavy Chain 1 (e.g., encoding a secondpolypeptide chain of a trispecific binding protein ofthe present disclosure) CD28supxCD3mid Light See above. SEQ ID Chain 1NO: 73 (e.g., encoding a first polypeptide chain of atrispecific binding protein of the present disclosure)mAb6 IgG1(knob) LALA CAGGTGCAGCTGGTGGAAAGCGGCGGAG SEQ IDP329A Heavy Chain 2 GCGTGGTGCAGCCTGGCAGGTCTCTGAGA NO: 82(e.g., encoding a third CTGAGCTGTGCCGCCAGCGGCTTCACCTTpolypeptide chain of a CAGCAGCTACGGAATGCACTGGGTGCGCtrispecific binding protein of CAGGCCCCTGGCAAAGGACTGGAATGGGthe present disclosure) TGGCCGTGATTTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCC GGTTCACCATCAGCGGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC TGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAATGTTCAGAGGCGCCTTCGA CTACTGGGGCCAGGGCACACTCGTGACCGTGTCTAGTGCGTCGACCAAGGGCCCCAGC GTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCT GCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCT GACCAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCAGCGGCCTGTACAGCCTG AGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACG TGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGC GACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCCGAAGCCGCCGGAGGCCCTTCCG TGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGT GACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTAC GTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCAAGAGAGGAACAGTACAA CAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGC AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGCCGCCCCCATCGAGAAAAC CATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCCCCATG CAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTGAAAGGCTTCTA CCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAA GACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTGACA GTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGC mAb6 Light2 See above. SEQ ID(e.g., encoding a fourth NO: 81 polypeptide chain of atrispecific binding protein of the present disclosure)mAb6xCD28supxCD3mid IgG1 NNSA CD28supxCD3mid See above. SEQ IDIgG1(hole) NNSA Heavy NO: 78 Chain 1 (e.g., encoding a secondpolypeptide chain of a trispecific binding protein ofthe present disclosure) CD28supxCD3mid Light See above. SEQ ID Chain 1NO: 73 (e.g., encoding a first polypeptide chain of atrispecific binding protein of the present disclosure)mAb6 IgG1(knob) NNSA CAGGTGCAGCTGGTGGAAAGCGGCGGAG SEQ ID Heavy Chain 2GCGTGGTGCAGCCTGGCAGGTCTCTGAGA NO: 83 (e.g., encoding a thirdCTGAGCTGTGCCGCCAGCGGCTTCACCTT polypeptide chain of aCAGCAGCTACGGAATGCACTGGGTGCGC trispecific binding protein ofCAGGCCCCTGGCAAAGGACTGGAATGGG the present disclosure)TGGCCGTGATTTGGTACGACGGCAGCAAC AAGTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGGCGACAACAGCAA GAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTAC TGCGCCAGAATGTTCAGAGGCGCCTTCGACTACTGGGGCCAGGGCACACTCGTGACCG TGTCTAGTGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAG CACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTG TCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAG CTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGT GGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCC AGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTATGTTGACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCGCGGGAGGAGCAGTACAACAATGCCTCCCGTGTGGTCAGCGTCCTCA CCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAATCAAGTCA GCCTGTGGTGCCTGGTAAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTACTCAAAACTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAA CGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCGGGT mAb6 Light2 See above.SEQ ID (e.g. , encoding a fourth NO: 81 polypeptide chain of atrispecific binding protein of the present disclosure)

CD38 Polypeptides

In some embodiments, a binding protein of the present disclosurecomprises an antigen binding site that binds an extracellular domain ofa human CD38 polypeptide and an extracellular domain of a cynomolgusmonkey CD38 polypeptide. Exemplary assays for determining whether anantigen binding site binds an antigen are described herein and known inthe art. In some embodiments, binding is determined by ELISA assay,e.g., as described infra. In some embodiments, binding is determined bySPR assay, e.g., as described infra. In some embodiments, binding isdetermined by flow cytometry assay using cells expressing a CD38polypeptide on their cell surface, e.g., as described infra. See, e.g.,Examples 1, 3, and 4.

In some embodiments, a binding protein of the present disclosure binds apurified polypeptide or fragment thereof comprising the amino acidsequence of SEQ ID NO:1 and/or 30 (e.g., as measured by ELISA or SPR).In some embodiments, a binding protein of the present disclosure binds apolypeptide or comprising the amino acid sequence of SEQ ID NO:1 and/or30 when expressed on the surface of a cell (e.g., as measured by flowcytometry).

In some embodiments, a binding protein of the present disclosure bindsto a CD38 isoform A polypeptide (e.g., comprising the amino acidsequence of SEQ ID NO:1). In some embodiments, a binding protein of thepresent disclosure binds to a CD38 isoform E polypeptide (e.g.,comprising the amino acid sequence of SEQ ID NO:105 and not comprisingthe full amino acid sequence of SEQ ID NO:1, consisting of the aminoacid sequence of SEQ ID NO:105, or consisting essentially of the aminoacid sequence of SEQ ID NO:105). In some embodiments, a binding proteinof the present disclosure binds to a CD38 isoform A polypeptide (e.g.,comprising the amino acid sequence of SEQ ID NO:1) and a CD38 isoform Epolypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:105and not comprising the full amino acid sequence of SEQ ID NO:1,consisting of the amino acid sequence of SEQ ID NO:105, or consistingessentially of the amino acid sequence of SEQ ID NO:105). Withoutwishing to be bound to theory, it is thought that binding to a CD38isoform E polypeptide can be advantageous, e.g., in targeting a bindingprotein of the present disclosure to cell(s) expressing a CD38 isoform Epolypeptide.

Human CD38 isoform A extracellular domain polypeptide sequence(SEQ ID NO: 1) RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDS SCTSEIHuman CD38 isoform E polypeptide sequence (SEQ ID NO: 105)RWRQQWSGPMTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSR RHFWECGSP

In some embodiments, the extracellular domain of a human CD38polypeptide comprises the amino acid sequence of SEQ ID NO:1. In someembodiments, the extracellular domain of a cynomolgus monkey CD38polypeptide comprises the amino acid sequence of SEQ ID NO:30.

Cynomolgus monkey CD38 polypeptide sequence (SEQ ID NO: 30)RWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTFEINYQSCPDWRKDCSNNPVSVFWKTVSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQALEAWVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKELQCVKNPEDS SCLSGI

Multispecific (e.g., Bispecific, Trispecific, or Multispecific) BindingProteins that Bind CD38 Polypeptides

In some embodiments, a binding protein of the present disclosure is abispecific binding protein comprising an antigen binding site that bindsone or more CD38 polypeptides and a second antigen binding site thatbinds a different target antigen. In some embodiments, a binding proteinof the present disclosure is a bispecific binding protein comprising anantigen binding site that binds one or more CD38 polypeptides and asecond antigen binding site that binds one or more CD38 polypeptides.

In some embodiments, a binding protein of the present disclosure is atrispecific binding protein comprising an antigen binding site thatbinds one or more CD38 polypeptides, a second antigen binding site, anda third antigen binding site. For example, in some embodiments, one ofthe antigen binding sites binds one or more CD38 polypeptide(s) (e.g.,the extracellular domain of a human and/or cynomolgus monkey CD38polypeptide), and one or two of the antigen binding sites binds a T-cellsurface protein. In some embodiments, one of the antigen binding sitesbinds one or more CD38 polypeptide(s) (e.g., the extracellular domain ofa human and/or cynomolgus monkey CD38 polypeptide), one of the antigenbinding sites binds a human CD3 polypeptide, and one of the antigenbinding sites binds a human CD28 polypeptide. Human CD3 and CD28polypeptides are known in the art. The amino acid sequences of exemplaryand non-limiting antibody variable domains that bind human CD3 and CD28polypeptides are provided herein.

In some embodiments, provided herein are binding proteins comprisingthree antigen binding sites that each bind one or more target proteins.In some embodiments, at least one of the three antigen binding sitesbinds an extracellular domain of a human CD38 polypeptide and anextracellular domain of a cynomolgus monkey CD38 polypeptide. In someembodiments, the human CD38 polypeptide comprises the amino acidsequence of SEQ ID NO:1, and/or the cynomolgus monkey CD38 polypeptidecomprises the amino acid sequence of SEQ ID NO:30. In some embodiments,the binding protein comprises an antigen binding site that binds anextracellular domain of a human CD38 polypeptide and an extracellulardomain of a cynomolgus monkey CD38 polypeptide and two antigen bindingsites that each bind a T-cell surface protein (e.g., a human CD28polypeptide and/or a human CD3 polypeptide).

In some embodiments, a binding protein of the present disclosure bindsone or more tumor target proteins (e.g., one or more CD38polypeptide(s)) and one or more T cell target proteins. In someembodiments, the binding protein is capable of binding one tumor targetprotein (e.g., one or more CD38 polypeptide(s)) and two differentepitopes on a single T cell target protein. In some embodiments, thebinding protein is capable of binding one tumor target protein (e.g.,one or more CD38 polypeptide(s)) and two different T cell targetproteins (e.g., CD28 and CD3). In some embodiments, the binding proteinis capable of binding one T cell target protein and two differentepitopes on a single tumor target protein (e.g., one or more CD38polypeptide(s)). In some embodiments, the binding protein is capable ofbinding one T cell target protein and two different tumor targetproteins (e.g., one or more CD38 polypeptide(s) and another tumor targetprotein). In some embodiments, the first and second polypeptide chainsof the binding protein form two antigen binding sites that target two Tcell target proteins, and the third and fourth polypeptide chains of thebinding protein form an antigen binding site that binds one or more CD38polypeptide(s). In some embodiments, the first and second polypeptidechains of the binding protein form two antigen binding sites that targettwo tumor target proteins (e.g., one or more CD38 polypeptide(s) andanother tumor target protein), and the third and fourth polypeptidechains of the binding protein form an antigen binding site that binds aT cell target protein. In some embodiments, the one or more T celltarget proteins are one or more of CD3 and CD28.

In some embodiments, the binding proteins specifically bind to one ormore CD38 polypeptide(s) and one or more target protein on a T-cellincluding a T cell receptor complex. These T-cell engager bindingproteins are capable of recruiting T cells transiently to target cellsand, at the same time, activating the cytolytic activity of the T cells.Examples of target proteins on T cells include but are not limited toCD3 and CD28, among others. Further examples of such antigen targets ortarget proteins are provided supra. In some embodiments, the trispecificbinding proteins may be generated by combining the antigen bindingdomains of two or more monospecific antibodies (parent antibodies) intoone antibody.

Bispecific Binding Protein Formats

In some embodiments, the binding protein of the disclosure is abispecific and/or bivalent binding protein comprising four polypeptidechains that form four antigen binding sites that bind one or more (e.g.,two) different antigen targets or target proteins (e.g., having astructure described in International Publication No. WO2012/135345). Insome embodiments, the binding protein is bivalent and/or bispecific. Insome embodiments, the binding protein is tetravalent and/ortetraspecific. In some embodiments, the binding protein is tetravalentand/or bispecific. In some embodiments, at least one of the antigenbinding sites binds a CD38 polypeptide (e.g., the extracellular domainof human and/or cynomolgus monkey CD38 polypeptides).

In some embodiments, the binding protein comprises two polypeptidechains having a structure represented by the formula:

V_(L1)-L₁-V_(L2)-L₂-C_(L)  [I]

and two polypeptide chains have a structure represented by the formula:

V_(H2)-L₃-V_(H1)-L₄-C_(H1)-Fc  [II]

wherein:V_(L1) is a first immunoglobulin light chain variable domain;V_(L2) is a second immunoglobulin light chain variable domain;V_(H1) is a first immunoglobulin heavy chain variable domain;V_(H2) is a second immunoglobulin heavy chain variable domain;C_(L) is an immunoglobulin light chain constant domain;C_(H1) is the immunoglobulin C_(H1) heavy chain constant domain;Fc comprises an immunoglobulin hinge region and C_(H2), C_(H3)immunoglobulin heavy chain constant domains;L₁, L₂, L₃, and L₄ are amino acid linkers;

-   and wherein the polypeptides of formula I and the polypeptides of    formula II form a cross-over light chain-heavy chain pair. In some    embodiments, V_(H1) and V_(L1) form an antigen binding domain that    binds a CD38 polypeptide, and V_(H2) and V_(L2) form an antigen    binding domain that binds another antigen target. In some    embodiments, V_(H2) and V_(L2) form an antigen binding domain that    binds a CD38 polypeptide, and V_(H1) and V_(L1) form an antigen    binding domain that binds another antigen target.

In some embodiments, the binding protein comprises two polypeptidechains that form two antigen binding sites, wherein a first polypeptidechain comprises

V_(L1)-L₁-V_(L2)-L₂-CL-Fc

and a second polypeptide chain comprises

V_(H2)-L₃-V_(H1)-L₄-C_(H1)-Fc

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

C_(H1) is the immunoglobulin CH1 heavy chain constant domain;

C_(H2) is an immunoglobulin C_(H2) heavy chain constant domain;

C_(H3) is an immunoglobulin C_(H3) heavy chain constant domain;

Fc comprises an immunoglobulin hinge region and C_(H2), C_(H3)immunoglobulin heavy chain constant domains; and

L₁, L₂, L₃, and L₄ are amino acid linkers;

-   -   wherein the first and second polypeptides form a cross-over        light chain-heavy chain pair. In some embodiments, V_(H1) and        V_(L1) form an antigen binding domain that binds a CD38        polypeptide, and V_(H2) and V_(L2) form an antigen binding        domain that binds another antigen target. In some embodiments,        V_(H2) and V_(L1) form an antigen binding domain that binds a        CD38 polypeptide, and V_(H1) and V_(L1) form an antigen binding        domain that binds another antigen target.

In some embodiments, the binding protein comprises three polypeptidechains that form two antigen binding sites, wherein a first polypeptidechain comprises

V_(L1)-L₁-V_(L2)-L₂-CL

a second polypeptide chain comprises

V_(H2)-L₃-V_(H1)-L₄-C_(H1)-Fc

a third polypeptide chain comprises an antibody Fc regionwherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

C_(H1) is the immunoglobulin C_(H1) heavy chain constant domain;

C_(H2) is an immunoglobulin C_(H2) heavy chain constant domain;

C_(H3) is an immunoglobulin C_(H3) heavy chain constant domain;

Fc comprises an immunoglobulin hinge region and C_(H2), C_(H3)immunoglobulin heavy chain constant domains; and

L₁, L₂, L₃, and L₄ are amino acid linkers;

wherein the first and second polypeptides form a cross-over lightchain-heavy chain pair. In some embodiments, V_(H1) m and V_(L1) form anantigen binding domain that binds a CD38 polypeptide, and V_(H2) andV_(L2) form an antigen binding domain that binds another antigen target.In some embodiments, V_(H2) and V_(L2) form an antigen binding domainthat binds a CD38 polypeptide, and V_(H1) and V_(L1) form an antigenbinding domain that binds another antigen target.

In some embodiments, the binding protein comprises a first polypeptidechain comprising a structure represented by the formula:

V_(L1)-L₁-V_(L2)-L₂-C_(L)  [I]

and a second polypeptide chain comprising a structure represented by theformula:

V_(H2)-L₃-V_(H1)-L₄-C_(H1)  [II]

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H1) is an immunoglobulin C_(H1) heavy chain constant domain; and

L₁, L₂, L₃ and L₄ are amino acid linkers;

-   -   wherein the polypeptide of formula I and the polypeptide of        formula II form a cross-over light chain-heavy chain pair. In        some embodiments, V_(H1) and V_(L1) form an antigen binding        domain that binds a CD38 polypeptide, and V_(H2) and V_(L2) form        an antigen binding domain that binds another antigen target. In        some embodiments, V_(H2) and V_(L2) form an antigen binding        domain that binds a CD38 polypeptide, and V_(H1) and V_(L1) form        an antigen binding domain that binds another antigen target.

In any of the bispecific binding proteins described supra, the targetantigen other than CD38 can be any of the following exemplary antigentargets: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC,B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5,CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (alsoknown as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known asMCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15(also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known asMIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2),CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK),CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (alsoknown as FCER2, a receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40,CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137(also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (alsoknown as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (alsoknown as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3),CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also knownas E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known asM-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1(also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3,DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1,FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2,HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B,IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R,IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (alsoknown as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35,ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2,MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, plateletreceptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptorfor IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2,TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known asa co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, andXCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more ofthe above antigen targets are human antigen targets.

In any of the bispecific binding proteins described supra, any linker orcombination of linkers described herein may be used. For example, insome embodiments, at least one of L₁, L₂, L₃ or L₄ is independently 0amino acids in length. In some embodiments, L₁, L₂, L₃ or L₄ are eachindependently at least one amino acid in length. In some embodiments,L₁, L₂, L₃ and L₄ each independently are zero amino acids in length orcomprise a sequence selected from the group consisting of GGGGSGGGGS(SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ IDNO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59). In someembodiments, L₁, L₂, L₃ and L₄ each independently comprise a sequenceselected from the group consisting of GGGGSGGGGS (SEQ ID NO:55),GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP(SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L₁comprises the sequence GQPKAAP (SEQ ID NO: 58), L₂ comprises thesequence TKGPS (SEQ ID NO:57), L₃ comprises the sequence S, and L₄comprises the sequence RT. In some embodiments, L₁ comprises thesequence GGGGSGGGGS (SEQ ID NO:55), L₂ comprises the sequence GGGGSGGGGS(SEQ ID NO:55), L₃ is 0 amino acids in length, and L₄ is 0 amino acidsin length. In some embodiments, L₁ comprises the sequence GGSGSSGSGG(SEQ ID NO:59), L₂ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₃is 0 amino acids in length, and L₄ is 0 amino acids in length. In someembodiments, L₁ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56),L₂ is 0 amino acids in length, L₃ comprises the sequence GGGGSGGGGSGGGGS(SEQ ID NO:56), and L₄ is 0 amino acids in length.

Trispecific Binding Proteins that Bind to CD38 Polypeptides

In some embodiments, the binding protein of the disclosure is atrispecific and/or trivalent binding protein comprising four polypeptidechains that form three antigen binding sites that bind one or more(e.g., three) different antigen targets or target proteins. In someembodiments, at least one of the antigen binding sites binds a CD38polypeptide (e.g., the extracellular domain of human and/or cynomolgusmonkey CD38 polypeptides). In some embodiments, a first polypeptidechain comprises a structure represented by the formula:

V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I]

and a second polypeptide chain comprises a structure represented by theformula:

V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3)  [II]

and a third polypeptide chain comprises a structure represented by theformula:

V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3)  [III]

and a fourth polypeptide chain comprises a structure represented by theformula:

V_(L3)-C_(L)  [IV]

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(L3) is a third immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

V_(H3) is a third immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H1) is an immunoglobulin C_(H1) heavy chain constant domain;

C_(H2) is an immunoglobulin C_(H2) heavy chain constant domain;

C_(H3) is an immunoglobulin C_(H3) heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the C_(H1) and C_(H2)domains; and

L₁, L₂, L₃ and L₄ are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula IIform a cross-over light chain-heavy chain pair.

In some embodiments, the binding protein of the disclosure is atrispecific and/or trivalent binding protein comprising four polypeptidechains that form three antigen binding sites that bind one or more (e.g.three) different antigen targets or target proteins. In someembodiments, at least one of the antigen binding sites binds a CD38polypeptide (e.g., the extracellular domain of human and/or cynomolgusmonkey CD38 polypeptides). In some embodiments, a first polypeptidechain comprises a structure represented by the formula:

V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I]

and a second polypeptide chain comprises a structure represented by theformula:

V_(H1)-L₃-V_(H2)-L-C_(H1)[II]

and a third polypeptide chain comprises a structure represented by theformula:

V_(H3)-C_(H1)  [III]

and a fourth polypeptide chain comprises a structure represented by theformula:

V_(L3)-C_(L)  [IV]

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(L3) is a third immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

V_(H3) is a third immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H1) is an immunoglobulin C_(H1) heavy chain constant domain; and

L₁, L₂, L₃ and L₄ are amino acid linkers;

-   and wherein the polypeptide of formula I and the polypeptide of    formula II form a cross-over light chain-heavy chain pair. In some    embodiments, the second and the third polypeptide chain further    comprise an Fc region linked to C_(H1), the Fc regions comprising an    immunoglobulin hinge region and C_(H2) and C_(H3) immunoglobulin    heavy chain constant domains.

In some embodiments, the first polypeptide chain and the secondpolypeptide chain have a cross-over orientation that forms two distinctantigen binding sites. In some embodiments, the VH1 and VL1 form abinding pair and form the first antigen binding site. In someembodiments, the VH2 and VL2 form a binding pair and form the secondantigen binding site. In some embodiments, the first antigen bindingsite binds a CD3 polypeptide (e.g., human CD3), and the second antigenbinding site binds a CD28 polypeptide (e.g., human CD28). In someembodiments, the second antigen binding site binds a CD3 polypeptide(e.g., human CD3), and the first antigen binding site binds a CD28polypeptide (e.g., human CD28). In some embodiments, the thirdpolypeptide and the fourth polypeptide form a third antigen bindingsite. In some embodiments, the VH3 and VL3 form a binding pair and formthe third antigen binding site. In some embodiments, the third antigenbinding site binds a CD38 polypeptide (e.g., human and optionallycynomolgus monkey CD38). Exemplary binding protein formats withcross-over orientations contemplated for use herein are also describedin U.S. patent application Ser. No. 15/487,243 and InternationalApplication No. PCT/US2017/027488.

In some embodiments in any of the bispecific, trispecific, ormultispecific binding proteins described herein, an antigen binding sitebinds a CD38 polypeptide (e.g., human and optionally cynomolgus monkeyCD38). In some embodiments, other (e.g., not binding to CD38) antigenbinding site(s) of any of the bispecific, trispecific, or multispecificbinding proteins described herein bind to CD28 or CD3. In someembodiments, the V_(H1) domain comprises a CDR-H1 sequence comprisingthe amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ IDNO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT(SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequencecomprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), andthe V_(L1) domain comprises a CDR-L1 sequence comprising the amino acidsequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), aCDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35)or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acidsequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H2)domain comprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) orIYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acidsequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L2) domaincomprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) orIYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acidsequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3) domaincomprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H1) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) orIYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acidsequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L1) domaincomprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H2) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) orIYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acidsequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L2) domaincomprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) orIYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acidsequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3) domaincomprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36).

In some embodiments, the V_(H1) domain comprises a CDR-H1 sequencecomprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42),and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY(SEQ ID NO:43), and/or the V_(L1) domain comprises a CDR-L1 sequencecomprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and aCDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ IDNO:46). In some embodiments, the V_(H2) domain comprises a CDR-H1sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41),a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ IDNO:42), and a CDR-H3 sequence comprising the amino acid sequence ofARMFRGAFDY (SEQ ID NO:43), and/or the V_(L2) domain comprises a CDR-L1sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), aCDR-L2 sequence comprising the amino acid sequence of AAS (SEQ IDNO:45), and a CDR-L3 sequence comprising the amino acid sequence ofLQDYIYYPT (SEQ ID NO:46). In some embodiments, the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acidsequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprisingthe amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and/or the V_(L3)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofQGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acidsequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising theamino acid sequence of LQDYIYYPT (SEQ ID NO:46).

In some embodiments, the V_(H1) domain comprises a CDR-H1 sequencecomprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42),and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY(SEQ ID NO:43), and the V_(L1) domain comprises a CDR-L1 sequencecomprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and aCDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ IDNO:46). In some embodiments, the V_(H2) domain comprises a CDR-H1sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41),a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ IDNO:42), and a CDR-H3 sequence comprising the amino acid sequence ofARMFRGAFDY (SEQ ID NO:43), and the V_(L2) domain comprises a CDR-L1sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), aCDR-L2 sequence comprising the amino acid sequence of AAS (SEQ IDNO:45), and a CDR-L3 sequence comprising the amino acid sequence ofLQDYIYYPT (SEQ ID NO:46). In some embodiments, the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acidsequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprisingthe amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the V_(L3)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofQGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acidsequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising theamino acid sequence of LQDYIYYPT (SEQ ID NO:46).

In some embodiments, the V_(H1) domain comprises a CDR-H1 sequencecomprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32),and a CDR-H3 sequence comprising the amino acid sequence ofARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_(L1) domain comprises aCDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ IDNO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQID NO:35), and a CDR-L3 sequence comprising the amino acid sequence ofQQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H1) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or theV_(L1) domain comprises a CDR-L1 sequence comprising the amino acidsequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising theamino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In someembodiments, the V_(H2) domain comprises a CDR-H1 sequence comprisingthe amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and aCDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQID NO:33), and/or the V_(L2) domain comprises a CDR-L1 sequencecomprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), aCDR-L2 sequence comprising the amino acid sequence of LAS (SEQ IDNO:35), and a CDR-L3 sequence comprising the amino acid sequence ofQQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H2) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or theV_(L2) domain comprises a CDR-L1 sequence comprising the amino acidsequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising theamino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In someembodiments, the V_(H3) domain comprises a CDR-H1 sequence comprisingthe amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and aCDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQID NO:33), and/or the V_(L3) domain comprises a CDR-L1 sequencecomprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), aCDR-L2 sequence comprising the amino acid sequence of LAS (SEQ IDNO:35), and a CDR-L3 sequence comprising the amino acid sequence ofQQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or theV_(L3) domain comprises a CDR-L1 sequence comprising the amino acidsequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising theamino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).

In some embodiments, the V_(H1) domain comprises a CDR-H1 sequencecomprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32),and a CDR-H3 sequence comprising the amino acid sequence ofARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L1) domain comprises a CDR-L1sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ IDNO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQID NO:35), and a CDR-L3 sequence comprising the amino acid sequence ofQQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H1) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L1)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofQSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acidsequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments,the V_(H2) domain comprises a CDR-H1 sequence comprising the amino acidsequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising theamino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequencecomprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), andthe V_(L2) domain comprises a CDR-L1 sequence comprising the amino acidsequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising theamino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In someembodiments, the V_(H2) domain comprises a CDR-H1 sequence comprisingthe amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequencecomprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and aCDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQID NO:33), and the V_(L2) domain comprises a CDR-L1 sequence comprisingthe amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequencecomprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).In some embodiments, the V_(H3) domain comprises a CDR-H1 sequencecomprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32),and a CDR-H3 sequence comprising the amino acid sequence ofARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3) domain comprises a CDR-L1sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ IDNO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQID NO:35), and a CDR-L3 sequence comprising the amino acid sequence ofQQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofQSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acidsequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36).

In some embodiments, the V_(H3) domain comprises a CDR-H1 sequencecomprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32),and a CDR-H3 sequence comprising the amino acid sequence ofARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_(L3) domain comprises aCDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ IDNO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQID NO:35), and a CDR-L3 sequence comprising the amino acid sequence ofQQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or theV_(L3) domain comprises a CDR-L1 sequence comprising the amino acidsequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising theamino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).

In some embodiments, the V_(H3) domain comprises the amino acid sequenceof SEQ ID NO:5, or the V_(L3) domain comprises the amino acid sequenceof SEQ ID NO:6. In some embodiments, the V_(H3) domain comprises theamino acid sequence of SEQ ID NO:17, or the V_(L3) domain comprises theamino acid sequence of SEQ ID NO:18. In some embodiments, the V_(H3)domain comprises the amino acid sequence of SEQ ID NO:21, or the V_(L3)domain comprises the amino acid sequence of SEQ ID NO:18. In someembodiments, the V_(H3) domain comprises the amino acid sequence of SEQID NO:23, or the V_(L3) domain comprises the amino acid sequence of SEQID NO:18. In some embodiments, the V_(H3) domain comprises the aminoacid sequence of SEQ ID NO:13, or the V_(L3) domain comprises the aminoacid sequence of SEQ ID NO:14.

In some embodiments, the V_(H3) domain comprises the amino acid sequenceof SEQ ID NO:5, and/or the V_(L3) domain comprises the amino acidsequence of SEQ ID NO:6. In some embodiments, the V_(H3) domaincomprises the amino acid sequence of SEQ ID NO:17, and the V_(L3) domaincomprises the amino acid sequence of SEQ ID NO:18. In some embodiments,the V_(H3) domain comprises the amino acid sequence of SEQ ID NO:21, andthe V_(L3) domain comprises the amino acid sequence of SEQ ID NO:18. Insome embodiments, the V_(H3) domain comprises the amino acid sequence ofSEQ ID NO:23, and the V_(L3) domain comprises the amino acid sequence ofSEQ ID NO:18. In some embodiments, the V_(H3) domain comprises the aminoacid sequence of SEQ ID NO:13, and the V_(L3) domain comprises the aminoacid sequence of SEQ ID NO:14.

In some embodiments, the V_(H3) domain comprises a CDR-H1 sequencecomprising the amino acid sequence of GFTFSSYG (SEQ ID NO:4), a CDR-H2sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42),and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY(SEQ ID NO:43), and/or the V_(L3) domain comprises a CDR-L1 sequencecomprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and aCDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ IDNO:46).

In some embodiments, the V_(H3) domain comprises the amino acid sequenceof SEQ ID NO:9, and/or the V_(L3) domain comprises the amino acidsequence of SEQ ID NO:10.

In some embodiments of any of the trispecific binding proteins of thepresent disclosure, one antigen binding domain binds to a CD3polypeptide (e.g., human CD3) and one antigen binding domain binds to aCD28 polypeptide (e.g., human CD28). In some embodiments, the V_(H1)domain comprises three CDRs from SEQ ID NOs:49 or 51 as shown in TableH, and the V_(L1) domain comprises three CDRs from SEQ ID NOs:50 or 52as shown in Table H. In some embodiments, the V_(H2) domain comprisesthree CDRs from SEQ ID NOs:49 or 51 as shown in Table H, and the V_(L2)domain comprises three CDRs from SEQ ID NOs:50 or 52 as shown in TableH. In some embodiments, the V_(H1) domain comprises three CDRs from SEQID NOs:53 or 84 as shown in Table H, and the V_(L1) domain comprisesthree CDRs from SEQ ID NOs:54 or 85 as shown in Table H. In someembodiments, the V_(H2) domain comprises three CDRs from SEQ ID NOs:53or 84 as shown in Table H, and the V_(L2) domain comprises three CDRsfrom SEQ ID NOs:54 or 85 as shown in Table H.

In some embodiments, the V_(H1) domain comprises the amino acid sequenceof SEQ ID NO:49, the V_(L1) domain comprises the amino acid sequence ofSEQ ID NO:50, the V_(H2) domain comprises the amino acid sequence of SEQID NO:53, and the V_(L2) domain comprises the amino acid sequence of SEQID NO:54. In some embodiments, the V_(H2) domain comprises the aminoacid sequence of SEQ ID NO:49, the V_(L2) domain comprises the aminoacid sequence of SEQ ID NO:50, the V_(H1) domain comprises the aminoacid sequence of SEQ ID NO:53, and the V_(L1) domain comprises the aminoacid sequence of SEQ ID NO:54. In some embodiments, the V_(H1) domaincomprises the amino acid sequence of SEQ ID NO:51, the V_(L1) domaincomprises the amino acid sequence of SEQ ID NO:52, the V_(H2) domaincomprises the amino acid sequence of SEQ ID NO:53, and the V_(L2) domaincomprises the amino acid sequence of SEQ ID NO:54. In some embodiments,the V_(H2) domain comprises the amino acid sequence of SEQ ID NO:51, theV_(L2) domain comprises the amino acid sequence of SEQ ID NO:52, theV_(H1) domain comprises the amino acid sequence of SEQ ID NO:53, and theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:54.

In some embodiments, the V_(H1) domain comprises the amino acid sequenceof SEQ ID NO:49, the V_(L1) domain comprises the amino acid sequence ofSEQ ID NO:50, the V_(H2) domain comprises the amino acid sequence of SEQID NO:53, the V_(L2) domain comprises the amino acid sequence of SEQ IDNO:54, the V_(H3) domain comprises the amino acid sequence of SEQ IDNO:13, and the V_(L3) domain comprises the amino acid sequence of SEQ IDNO:14. In some embodiments, the V_(H1) domain comprises the amino acidsequence of SEQ ID NO:49, the V_(L1) domain comprises the amino acidsequence of SEQ ID NO:50, the V_(H2) domain comprises the amino acidsequence of SEQ ID NO:53, the V_(L2) domain comprises the amino acidsequence of SEQ ID NO:54, the V_(H3) domain comprises the amino acidsequence of SEQ ID NO:9, and the V_(L3) domain comprises the amino acidsequence of SEQ ID NO:10.

In certain embodiments, the first polypeptide chain comprises apolypeptide sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO:61, the second polypeptide chain comprises apolypeptide sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO:60, the third polypeptide chain comprises apolypeptide sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO:62, and the fourth polypeptide chain comprises apolypeptide sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO:63. In certain embodiments, the first polypeptidechain comprises a polypeptide sequence that is at least 95% identical tothe amino acid sequence of SEQ ID NO:61, the second polypeptide chaincomprises a polypeptide sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:64, the third polypeptide chaincomprises a polypeptide sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:65, and the fourth polypeptide chaincomprises a polypeptide sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:63. In certain embodiments, the firstpolypeptide chain comprises a polypeptide sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:61, the secondpolypeptide chain comprises a polypeptide sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:66, the thirdpolypeptide chain comprises a polypeptide sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:67, and the fourthpolypeptide chain comprises a polypeptide sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:63. In certainembodiments, the first polypeptide chain comprises a polypeptidesequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:61, the second polypeptide chain comprises a polypeptidesequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:60, the third polypeptide chain comprises a polypeptidesequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:68, and the fourth polypeptide chain comprises a polypeptidesequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:69. In certain embodiments, the first polypeptide chaincomprises a polypeptide sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:61, the second polypeptide chaincomprises a polypeptide sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:64, the third polypeptide chaincomprises a polypeptide sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:70, and the fourth polypeptide chaincomprises a polypeptide sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:69. In certain embodiments, the firstpolypeptide chain comprises a polypeptide sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:61, the secondpolypeptide chain comprises a polypeptide sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:66, the thirdpolypeptide chain comprises a polypeptide sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:71, and the fourthpolypeptide chain comprises a polypeptide sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:69.

In certain embodiments, the first polypeptide chain comprises the aminoacid sequence of SEQ ID NO:61, the second polypeptide chain comprisesthe amino acid sequence of SEQ ID NO:60, the third polypeptide chaincomprises the amino acid sequence of SEQ ID NO:62, and the fourthpolypeptide chain comprises the amino acid sequence of SEQ ID NO:63. Incertain embodiments, the first polypeptide chain comprises the aminoacid sequence of SEQ ID NO:61, the second polypeptide chain comprisesthe amino acid sequence of SEQ ID NO:64, the third polypeptide chaincomprises the amino acid sequence of SEQ ID NO:65, and the fourthpolypeptide chain comprises the amino acid sequence of SEQ ID NO:63. Incertain embodiments, the first polypeptide chain comprises the aminoacid sequence of SEQ ID NO:61, the second polypeptide chain comprisesthe amino acid sequence of SEQ ID NO:66, the third polypeptide chaincomprises the amino acid sequence of SEQ ID NO:67, and the fourthpolypeptide chain comprises the amino acid sequence of SEQ ID NO:63. Incertain embodiments, the first polypeptide chain comprises the aminoacid sequence of SEQ ID NO:61, the second poly peptide chain comprisesthe amino acid sequence of SEQ ID NO:60, the third polypeptide chaincomprises the amino acid sequence of SEQ ID NO:68, and the fourthpolypeptide chain comprises the amino acid sequence of SEQ ID NO:69. Incertain embodiments, the first polypeptide chain comprises the aminoacid sequence of SEQ ID NO:61, the second polypeptide chain comprisesthe amino acid sequence of SEQ ID NO:64, the third polypeptide chaincomprises the amino acid sequence of SEQ ID NO:70, and the fourthpolypeptide chain comprises the amino acid sequence of SEQ ID NO:69. Incertain embodiments, the first polypeptide chain comprises the aminoacid sequence of SEQ ID NO:61, the second polypeptide chain comprisesthe amino acid sequence of SEQ ID NO:66, the third polypeptide chaincomprises the amino acid sequence of SEQ ID NO:71, and the fourthpolypeptide chain comprises the amino acid sequence of SEQ ID NO:69.

In any of the trispecific binding proteins described supra, the targetantigen other than CD38 can be any of the following exemplary antigentargets: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC,B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5,CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (alsoknown as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known asMCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15(also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known asMIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2),CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK),CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (alsoknown as FCER2, a receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40,CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137(also known as 41 BB), CD137L, CD152 (also known as CTLA4), CD154 (alsoknown as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (alsoknown as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3),CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also knownas E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known asM-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1(also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3,DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1,FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2,HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B,IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R,IL10, rhIL10, IL12, IL13, IL3Ra1, IL13Ra2, IL15, IL17, IL17Rb (alsoknown as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35,ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2,MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, plateletreceptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptorfor IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2,TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known asa co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, andXCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more ofthe above antigen targets are human antigen targets.

In some embodiments, a binding protein of the present disclosure is anantibody. In some embodiments, the antibody is a monoclonal antibody. Insome embodiments, the antibody is a chimeric, humanized, or humanantibody.

The binding proteins of the disclosure may be prepared using domains orsequences obtained or derived from any human or non-human antibody,including, for example, human, murine, or humanized antibodies.

Linkers

In some embodiments, the linkers L₁, L₂, L₃ and L₄ range from no aminoacids (length=0) to about 100 amino acids long, or less than 100, 50,40, 30, 20, or 15 amino acids or less. The linkers can also be 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 amino acids long. L₁, L₂, L₃ and L₄ in onebinding protein may all have the same amino acid sequence or may allhave different amino acid sequences.

Examples of suitable linkers include a single glycine (Gly) residue; adiglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide withfour glycine residues; a peptide with five glycine residues; a peptidewith six glycine residues; a peptide with seven glycine residues; and apeptide with eight glycine residues. Other combinations of amino acidresidues may be used such as the peptide GGGGSGGGGS (SEQ ID NO: 55), thepeptide GGGGSGGGGSGGGGS (SEQ ID NO: 56), the peptide TKGPS (SEQ ID NO:57), the peptide GQPKAAP (SEQ ID NO:58), and the peptide GGSGSSGSGG (SEQID NO:59). The examples listed above are not intended to limit the scopeof the disclosure in any way, and linkers comprising randomly selectedamino acids selected from the group consisting of valine, leucine,isoleucine, serine, threonine, lysine, arginine, histidine, aspartate,glutamate, asparagine, glutamine, glycine, and proline have been shownto be suitable in the binding proteins. For additional descriptions oflinker sequences, see, e.g., WO2012135345 and International ApplicationNo. PCT/US2017/027488.

The identity and sequence of amino acid residues in the linker may varydepending on the type of secondary structural element necessary toachieve in the linker. For example, glycine, serine, and alanine arebest for linkers having maximum flexibility. Some combination ofglycine, proline, threonine, and serine are useful if a more rigid andextended linker is necessary. Any amino acid residue may be consideredas a linker in combination with other amino acid residues to constructlarger peptide linkers as necessary depending on the desired properties.

In some embodiments, at least one of L₁, L₂, L₃ or L₄ is independently 0amino acids in length. In some embodiments, L₁, L₂, L₃ or L₄ are eachindependently at least one amino acid in length. In some embodiments,the length of L₁ is at least twice the length of L₃. In someembodiments, the length of L₂ is at least twice the length of L₄. Insome embodiments, the length of L₁ is at least twice the length of L₃,and the length of L₂ is at least twice the length of L₄. In someembodiments, L₁ is 3 to 12 amino acid residues in length, L₂ is 3 to 14amino acid residues in length, L₃ is 1 to 8 amino acid residues inlength, and L₄ is 1 to 3 amino acid residues in length. In someembodiments, L₁ is 5 to 10 amino acid residues in length, L₂ is 5 to 8amino acid residues in length, L₃ is 1 to 5 amino acid residues inlength, and L₄ is 1 to 2 amino acid residues in length. In someembodiments, L₁ is 7 amino acid residues in length, L₂ is 5 amino acidresidues in length, L₃ is 1 amino acid residue in length, and L₄ is 2amino acid residues in length. In some embodiments, L₁ is 10 amino acidresidues in length, L₂ is 10 amino acid residues in length, L₃ is 0amino acid residue in length, and L₄ is 0 amino acid residues in length.In some embodiments, L₁, L₂, L₃, and L₄ each have an independentlyselected length from 0 to 15 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 amino acids), wherein at least two ofthe linkers have a length of 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). In some embodiments,L₁, L₂, L₃, and L₄ are each 0 amino acids in length.

In some embodiments, L₁, L₂, L₃, and/or L₄ comprise a sequence derivedfrom a naturally occurring sequence at the junction between an antibodyvariable domain and an antibody constant domain (e.g., as described inWO2012/135345). For example, in some embodiments, the linker comprises asequence found at the transition between an endogenous V_(H1) and C_(H1)domain, or between an endogenous V_(L) and C_(L) domain (e.g., kappa orlambda). In some embodiments, the linker comprises a sequence found atthe transition between an endogenous human V_(H) and C_(H1) domain, orbetween an endogenous human V_(L) and C_(L) domain (e.g., human kappa orlambda).

In some embodiments, L₁, L₂, L₃ and L₄ each independently are zero aminoacids in length or comprise a sequence selected from the groupconsisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56),S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG(SEQ ID NO:59). In some embodiments, L₁, L₂, L₃ and L₄ eachindependently comprise a sequence selected from the group consisting ofGGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS(SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59).

In some embodiments, L₁ comprises the sequence GQPKAAP (SEQ ID NO: 58),L₂ comprises the sequence TKGPS (SEQ ID NO:57), L₃ comprises thesequence S, and L₄ comprises the sequence RT. In some embodiments, L₁comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₂ comprises thesequence GGGGSGGGGS (SEQ ID NO:55), L₃ is 0 amino acids in length, andL₄ is 0 amino acids in length. In some embodiments, L₁ comprises thesequence GGSGSSGSGG (SEQ ID NO:59), L₂ comprises the sequence GGSGSSGSGG(SEQ ID NO:59), L₃ is 0 amino acids in length, and L₄ is 0 amino acidsin length. In some embodiments, L₁ comprises the sequenceGGGGSGGGGSGGGGS (SEQ ID NO:56), L₂ is 0 amino acids in length, L₃comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and L₄ is 0 aminoacids in length.

Fc Regions and Constant Domains

In some embodiments, a binding protein of the present disclosurecomprises a full-length antibody heavy chain or a polypeptide chaincomprising an Fc region. In some embodiments, the Fc region is a humanFc region, e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region. In someembodiments, the Fc region includes an antibody hinge, C_(H1), C_(H2),C_(H3), and optionally C_(H4) domains. In some embodiments, the Fcregion is a human IgG1 Fc region. In some embodiments, the Fc region isa human IgG4 Fc region. In some embodiments, the Fc region includes oneor more of the mutations described infra.

In some embodiments, a binding protein of the present disclosureincludes one or two Fc variants. The term “Fc variant” as used hereinrefers to a molecule or sequence that is modified from a native Fc butstill comprises a binding site for the salvage receptor, FcRn (neonatalFc receptor). Exemplary Fc variants, and their interaction with thesalvage receptor, are known in the art. Thus, the term “Fc variant” cancomprise a molecule or sequence that is humanized from a non-humannative Fc. Furthermore, a native Fc comprises regions that can beremoved because they provide structural features or biological activitythat are not required for the antibody-like binding proteins of theinvention. Thus, the term “Fc variant” comprises a molecule or sequencethat lacks one or more native Fc sites or residues, or in which one ormore Fc sites or residues has be modified, that affect or are involvedin: (1) disulfide bond formation, (2) incompatibility with a selectedhost cell, (3) N-terminal heterogeneity upon expression in a selectedhost cell, (4) glycosylation, (5) interaction with complement, (6)binding to an Fc receptor other than a salvage receptor, or (7)antibody-dependent cellular cytotoxicity (ADCC).

In some embodiments, the Fc region comprises one or more mutations thatreduce or eliminate Fc receptor binding and/or effector function of theFc region (e.g., Fc receptor-mediated antibody-dependent cellularphagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/orantibody-dependent cellular cytotoxicity (ADCC)).

In some embodiments, the Fc region is a human IgG1 Fc region comprisingone or more amino acid substitutions at positions corresponding topositions 234, 235, and/or 329 of human IgG1 according to EU Index. Insome embodiments, the amino acid substitutions are L234A, L235A, and/orP329A. In some embodiments, the Fc region is a human IgG1 Fc regioncomprising amino acid substitutions at positions corresponding topositions 298, 299, and/or 300 of human IgG1 according to EU Index. Insome embodiments, the amino acid substitutions are S298N, T299A, and/orY300S.

In some embodiments, the Fc region is a human IgG4 Fc region comprisingone or more mutations that reduce or eliminate FcγI and/or FcγIIbinding. In some embodiments, the Fc region is a human IgG4 Fc regioncomprising one or more mutations that reduce or eliminate FcγI and/orFcγII binding but do not affect FcRn binding. In some embodiments, theFc region is a human IgG4 Fc region comprising amino acid substitutionsat positions corresponding to positions 228 and/or 409 of human IgG4according to EU Index. In some embodiments, the amino acid substitutionsare S228P and/or R409K. In some embodiments, the Fc region is a humanIgG4 Fc region comprising amino acid substitutions at positionscorresponding to positions 234 and/or 235 of human IgG4 according to EUIndex. In some embodiments, the amino acid substitutions are F234Aand/or L235A. In some embodiments, the Fc region is a human IgG4 Fcregion comprising amino acid substitutions at positions corresponding topositions 228, 234, 235, and/or 409 of human IgG4 according to EU Index.In some embodiments, the amino acid substitutions are S228P, F234A,L235A, and/or R409K. In some embodiments, the Fc region is a human IgG4Fc region comprising amino acid substitutions at positions correspondingto positions 233-236 of human IgG4 according to EU Index. In someembodiments, the amino acid substitutions are E233P, F234V, L235A, and adeletion at 236. In some embodiments, the Fc region is a human IgG4 Fcregion comprising amino acid mutations at substitutions corresponding topositions 228, 233-236, and/or 409 of human IgG4 according to EU Index.In some embodiments, the amino acid mutations are S228P; E233P, F234V,L235A, and a deletion at 236; and/or R409K.

In some embodiments, a binding protein of the present disclosurecomprises one or more mutations to improve purification, e.g., bymodulating the affinity for a purification reagent. For example, it isknown that heterodimeric binding proteins can be selectively purifiedaway from their homodimeric forms if one of the two Fc regions of theheterodimeric form contains mutation(s) that reduce or eliminate bindingto Protein A, because the heterodimeric form will have an intermediateaffinity for Protein A-based purification than either homodimeric formand can be selectively eluted from Protein A, e.g., by use of adifferent pH (See e.g., Smith, E. J. et al. (2015) Sci. Rep. 5:17943).In some embodiments, the mutation comprises substitutions at positionscorresponding to positions 435 and 436 of human IgG1 or IgG4 accordingto EU Index, wherein the amino acid substitutions are H435R and Y436F.In some embodiments, the binding protein comprises a second polypeptidechain further comprising a first Fc region linked to C_(H1), the firstFc region comprising an immunoglobulin hinge region and C_(H2) andC_(H3) immunoglobulin heavy chain constant domains, and a thirdpolypeptide chain further comprising a second Fc region linked toC_(H1), the second Fc region comprising an immunoglobulin hinge regionand C_(H2) and C_(H3) immunoglobulin heavy chain constant domains; andwherein only one of the first and the second Fc regions comprises aminoacid substitutions at positions corresponding to positions 435 and 436of human IgG1 or IgG4 according to EU Index, wherein the amino acidsubstitutions are H435R and Y436F. In some embodiments, a bindingprotein of the present disclosure comprises knob and hole mutations andone or more mutations to improve purification. In some embodiments, thefirst and/or second Fc regions are human IgG1 Fc regions. In someembodiments, the first and/or second Fc regions are human IgG4 Fcregions.

To improve the yields of some binding proteins (e.g., bispecific ortrispecific binding proteins), the C_(H3) domains can be altered by the“knob-into-holes” technology which is described in detail with severalexamples in, for example, International Publication No. WO 96/027011,Ridgway et al., 1996, Protein Eng. 9: 617-21; and Merchant et al., 1998,Nat. Biotechnol. 16: 677-81. Specifically, the interaction surfaces ofthe two C_(H3) domains are altered to increase the heterodimerisation ofboth heavy chains containing these two C_(H3) domains. Each of the twoC_(H3) domains (of the two heavy chains) can be the “knob,” while theother is the “hole.” The introduction of a disulfide bridge furtherstabilizes the heterodimers (Merchant et al., 1998; Atwell et al., 1997,J. Mol. Biol. 270: 26-35) and increases the yield. In particularembodiments, the knob is on the second pair of polypeptides with asingle variable domain. In other embodiments, the knob is on the firstpair of polypeptides having the cross-over orientation. In yet otherembodiments, the C_(H3) domains do not include a knob in hole.

In some embodiments, a binding protein of the present disclosure (e.g.,a trispecific binding protein) comprises a “knob” mutation on the secondpolypeptide chain and a “hole” mutation on the third polypeptide chain.In some embodiments, a binding protein of the present disclosurecomprises a “knob” mutation on the third polypeptide chain and a “hole”mutation on the second polypeptide chain. In some embodiments, the“knob” mutation comprises substitution(s) at positions corresponding topositions 354 and/or 366 of human IgG1 or IgG4 according to EU Index. Insome embodiments, the amino acid substitutions are S354C, T366W, T366Y,S354C and T366W, or S354C and T366Y. In some embodiments, the “knob”mutation comprises substitutions at positions corresponding to positions354 and 366 of human IgG1 or IgG4 according to EU Index. In someembodiments, the amino acid substitutions are S354C and T366W. In someembodiments, the “hole” mutation comprises substitution(s) at positionscorresponding to positions 407 and, optionally, 349, 366, and/or 368 andof human IgG1 or IgG4 according to EU Index. In some embodiments, theamino acid substitutions are Y407V or Y407T and optionally Y349C, T366S,and/or L368A. In some embodiments, the “hole” mutation comprisessubstitutions at positions corresponding to positions 349, 366, 368, and407 of human IgG1 or IgG4 according to EU Index. In some embodiments,the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

In some embodiments, the second polypeptide chain further comprises afirst Fc region linked to CH1, the first Fc region comprising animmunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chainconstant domains, wherein the first Fc region comprises amino acidsubstitution(s) at positions corresponding to positions 366 andoptionally 354 of human IgG1 or IgG4 according to EU Index, wherein theamino acid substitutions are T366W or T366Y and optionally S354C; andwherein the third polypeptide chain further comprises a second Fc regionlinked to CH1, the second Fc region comprising an immunoglobulin hingeregion and CH2 and CH3 immunoglobulin heavy chain constant domains,wherein the second Fc region comprises amino acid substitution(s) atpositions corresponding to positions 407 and optionally 349, 366, and/or368 and of human IgG1 or IgG4 according to EU Index, wherein the aminoacid substitutions are Y407V or Y407T and optionally Y349C, T366S,and/or L368A.

In some embodiments, the second polypeptide chain further comprises afirst Fc region linked to CH1, the first Fc region comprising animmunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chainconstant domains, wherein the first Fc region comprises amino acidsubstitution(s) at positions corresponding to positions 407 andoptionally 349, 366, and/or 368 and of human IgG1 or IgG4 according toEU Index, wherein the amino acid substitutions are Y407V or Y407T andoptionally Y349C, T366S, and/or L368A; and wherein the third polypeptidechain further comprises a second Fc region linked to CH1, the second Fcregion comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the second Fcregion comprises amino acid substitution(s) at positions correspondingto positions 366 and optionally 354 of human IgG1 or IgG4 according toEU Index, wherein the amino acid substitutions are T366W or T366Y andoptionally S354C.

In some embodiments, the second polypeptide chain further comprises afirst Fc region linked to CH1, the first Fc region comprising animmunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chainconstant domains, wherein the first Fc region comprises amino acidsubstitution at position corresponding to position 366 of human IgG1 orIgG4 according to EU Index, wherein the amino acid substitution isT366W; and wherein the third polypeptide chain further comprises asecond Fc region linked to CH1, the second Fc region comprising animmunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chainconstant domains, wherein the second Fc region comprises amino acidsubstitution(s) at positions corresponding to positions 366, 368, and/or407 and of human IgG1 or IgG4 according to EU Index, wherein the aminoacid substitutions are T366S, L368A, and/or Y407V.

In some embodiments, the second polypeptide chain further comprises afirst Fc region linked to CH1, the first Fc region comprising animmunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chainconstant domains, wherein the first Fc region comprises amino acidsubstitution(s) at positions corresponding to positions 366, 368, and/or407 and of human IgG1 or IgG4 according to EU Index, wherein the aminoacid substitutions are T366S, L368A, and/or Y407V; and wherein the thirdpolypeptide chain further comprises a second Fc region linked to CH1,the second Fc region comprising an immunoglobulin hinge region and CH2and CH3 immunoglobulin heavy chain constant domains, wherein the secondFc region comprises amino acid substitution at position corresponding toposition 366 of human IgG1 or IgG4 according to EU Index, wherein theamino acid substitution is T366W.

In some embodiments, the second polypeptide chain further comprises afirst Fc region linked to CH1, the first Fc region comprising animmunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chainconstant domains, wherein the first Fc region comprises amino acidsubstitutions at positions corresponding to positions 354 and 366 ofhuman IgG1 or IgG4 according to EU Index, wherein the amino acidsubstitutions are S354C and T366W; and wherein the third polypeptidechain further comprises a second Fc region linked to CH1, the second Fcregion comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the second Fcregion comprises amino acid substitutions at positions corresponding topositions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EUIndex, wherein the amino acid substitutions are Y349C, T366S, L368A, andY407V. In some embodiments, the second polypeptide chain furthercomprises a first Fc region linked to CH1, the first Fc regioncomprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulinheavy chain constant domains, wherein the first Fc region comprisesamino acid substitutions at positions corresponding to positions 349,366, 368, and 407 of human IgG1 or IgG4 according to EU Index, whereinthe amino acid substitutions are Y349C, T366S, L368A, and Y407V; andwherein the third polypeptide chain further comprises a second Fc regionlinked to CH1, the second Fc region comprising an immunoglobulin hingeregion and CH2 and CH3 immunoglobulin heavy chain constant domains,wherein the second Fc region comprises amino acid substitutions atpositions corresponding to positions 354 and 366 of human IgG1 or IgG4according to EU Index, wherein the amino acid substitutions are S354Cand T366W. In some embodiments, the first and/or second Fc regions arehuman IgG1 Fc regions. In some embodiments, the first and/or second Fcregions are human IgG4 Fc regions.

In some embodiments, the second polypeptide chain further comprises afirst Fc region linked to CH1, wherein the first Fc region is a humanIgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the first Fc regioncomprises amino acid substitutions at positions corresponding topositions 228, 354, 366, and 409 of human IgG4 according to EU Index,wherein the amino acid substitutions are S228P, S354C, T366W, and R409K;and wherein the third polypeptide chain further comprises a second Fcregion linked to CH1, wherein the second Fc region is a human IgG4 Fcregion comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the second Fcregion comprises amino acid substitutions at positions corresponding topositions 228, 349, 366, 368, 407, and 409 of human IgG4 according to EUIndex, wherein the amino acid substitutions are S228P, Y349C, T366S,L368A, Y407V, and R409K. In some embodiments, the second polypeptidechain further comprises a first Fc region linked to CH1, wherein thefirst Fc region is a human IgG4 Fc region comprising an immunoglobulinhinge region and CH2 and CH3 immunoglobulin heavy chain constantdomains, wherein the first Fc region comprises amino acid substitutionsat positions corresponding to positions 228, 349, 366, 368, 407, and 409of human IgG4 according to EU Index, wherein the amino acidsubstitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; andwherein the third polypeptide chain further comprises a second Fc regionlinked to CH1, wherein the second Fc region is a human IgG4 Fc regioncomprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulinheavy chain constant domains, wherein the second Fc region comprisesamino acid substitutions at positions corresponding to positions 228,354, 366, and 409 of human IgG4 according to EU Index, wherein the aminoacid substitutions are S228P, S354C, T366W, and R409K.

In some embodiments, the second polypeptide chain further comprises afirst Fc region linked to CH1, wherein the first Fc region is a humanIgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the first Fc regioncomprises amino acid substitutions at positions corresponding topositions 234, 235, 354, and 366 of human IgG4 according to EU Index,wherein the amino acid substitutions are F234A. L235A, S354C, and T366W;and wherein the third polypeptide chain further comprises a second Fcregion linked to CH1, wherein the second Fc region is a human IgG4 Fcregion comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the second Fcregion comprises amino acid substitutions at positions corresponding topositions 234, 235, 349, 366, 368, and 407 of human IgG4 according to EUIndex, wherein the amino acid substitutions are F234A, L235A, Y349C,T366S, L368A, and Y407V. In some embodiments, the second polypeptidechain further comprises a first Fc region linked to CH1, wherein thefirst Fc region is a human IgG4 Fc region comprising an immunoglobulinhinge region and CH2 and CH3 immunoglobulin heavy chain constantdomains, wherein the first Fc region comprises amino acid substitutionsat positions corresponding to positions 234, 235, 349, 366, 368, and 407of human IgG4 according to EU Index, wherein the amino acidsubstitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V; andwherein the third polypeptide chain further comprises a second Fc regionlinked to CH1, wherein the second Fc region is a human IgG4 Fc regioncomprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulinheavy chain constant domains, wherein the second Fc region comprisesamino acid substitutions at positions corresponding to positions 234,235, 354, and 366 of human IgG4 according to EU Index, wherein the aminoacid substitutions are F234A, L235A, S354C, and T366W.

In some embodiments, the second polypeptide chain further comprises afirst Fc region linked to CH1, wherein the first Fc region is a humanIgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the first Fc regioncomprises amino acid substitutions at positions corresponding topositions 228, 234, 235, 354, 366, and 409 of human IgG4 according to EUIndex, wherein the amino acid substitutions are S228P, F234A, L235A,S354C, T366W, and R409K; and wherein the third polypeptide chain furthercomprises a second Fc region linked to CH1, wherein the second Fc regionis a human IgG4 Fc region comprising an immunoglobulin hinge region andCH2 and CH3 immunoglobulin heavy chain constant domains, wherein thesecond Fc region comprises amino acid substitutions at positionscorresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 ofhuman IgG4 according to EU Index, wherein the amino acid substitutionsare S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In someembodiments, the second polypeptide chain further comprises a first Fcregion linked to CH1, wherein the first Fc region is a human IgG4 Fcregion comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the first Fc regioncomprises amino acid substitutions at positions corresponding topositions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4according to EU Index, wherein the amino acid substitutions are S228P,F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and wherein thethird polypeptide chain further comprises a second Fc region linked toCH1, wherein the second Fc region is a human IgG4 Fc region comprisingan immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavychain constant domains, wherein the second Fc region comprises aminoacid substitutions at positions corresponding to positions 228, 234,235, 354, 366, and 409 of human IgG4 according to EU Index, wherein theamino acid substitutions are S228P, F234A, L235A, S354C, T366W, andR409K.

In some embodiments, a binding protein of the present disclosurecomprises one or more mutations to improve serum half-life (See e.g.,Hinton, P. R. et al. (2006) J. Immunol. 176(1):346-56). In someembodiments, the mutation comprises substitutions at positionscorresponding to positions 428 and 434 of human IgG1 or IgG4 accordingto EU Index, wherein the amino acid substitutions are M428L and N434S.In some embodiments, the binding protein comprises a second polypeptidechain further comprising a first Fc region linked to CH1, the first Fcregion comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, and a third polypeptidechain further comprising a second Fc region linked to CH1, the second Fcregion comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the first and/orsecond Fc regions comprise amino acid substitutions at positionscorresponding to positions 428 and 434 of human IgG1 or IgG4 accordingto EU Index, wherein the amino acid substitutions are M428L and N434S.In some embodiments, a binding protein of the present disclosurecomprises knob and hole mutations and one or more mutations to improveserum half-life. In some embodiments, the first and/or second Fc regionsare human IgG1 Fc regions. In some embodiments, the first and/or secondFc regions are human IgG4 Fc regions.

In some embodiments, a binding protein of the present disclosurecomprises one or more mutations to improve stability, e.g., of the hingeregion and/or dimer interface of IgG4 (See e.g., Spiess, C. et al.(2013) J. Biol. Chem. 288:26583-26593). In some embodiments, themutation comprises substitutions at positions corresponding to positions228 and 409 of human IgG4 according to EU Index, wherein the amino acidsubstitutions are S228P and R409K. In some embodiments, the bindingprotein comprises a second polypeptide chain further comprising a firstFc region linked to C_(H1), the first Fc region comprising animmunoglobulin hinge region and C_(H2) and C_(H), immunoglobulin heavychain constant domains, and a third polypeptide chain further comprisinga second Fc region linked to C_(H1), the second Fc region comprising animmunoglobulin hinge region and C_(H2) and C_(H3) immunoglobulin heavychain constant domains; wherein the first and second Fc regions arehuman IgG4 Fc regions; and wherein the first and the second Fc regionseach comprise amino acid substitutions at positions corresponding topositions 228 and 409 of human IgG4 according to EU Index, wherein theamino acid substitutions are S228P and R409K. In some embodiments, abinding protein of the present disclosure comprises knob and holemutations and one or more mutations to improve stability. In someembodiments, the first and/or second Fc regions are human IgG4 Fcregions.

In some embodiments, a binding protein of the present disclosurecomprises one or more mutations to improve purification, e.g., bymodulating the affinity for a purification reagent. For example, it isknown that heterodimeric binding proteins can be selectively purifiedaway from their homodimeric forms if one of the two Fc regions of theheterodimeric form contains mutation(s) that reduce or eliminate bindingto Protein A, because the heterodimeric form will have an intermediateaffinity for Protein A-based purification than either homodimeric formand can be selectively eluted from Protein A, e.g., by use of adifferent pH (See e.g., Smith, E. J. et al. (2015) Sci. Rep. 5:17943).In some embodiments, the mutation comprises substitutions at positionscorresponding to positions 435 and 436 of human IgG1 or IgG4 accordingto EU Index, wherein the amino acid substitutions are H435R and Y436F.In some embodiments, the binding protein comprises a second polypeptidechain further comprising a first Fc region linked to C_(H1), the firstFc region comprising an immunoglobulin hinge region and C_(H2) andC_(H3) immunoglobulin heavy chain constant domains, and a thirdpolypeptide chain further comprising a second Fc region linked toC_(H1), the second Fc region comprising an immunoglobulin hinge regionand C_(H2) and C_(H3) immunoglobulin heavy chain constant domains; andwherein only one of the first and the second Fc regions comprises aminoacid substitutions at positions corresponding to positions 435 and 436of human IgG1 or IgG4 according to EU Index, wherein the amino acidsubstitutions are H435R and Y436F. In some embodiments, a bindingprotein of the present disclosure comprises knob and hole mutations andone or more mutations to improve purification. In some embodiments, thefirst and/or second Fc regions are human IgG1 Fc regions. In someembodiments, the first and/or second Fc regions are human IgG4 Fcregions.

In some embodiments, a binding protein of the present disclosurecomprises one or more mutations to improve serum half-life (See e.g.,Hinton, P. R. et al. (2006) J. Immunol. 176(1):346-56). In someembodiments, the mutation comprises substitutions at positionscorresponding to positions 428 and 434 of human IgG1 or IgG4 accordingto EU Index, wherein the amino acid substitutions are M428L and N434S.In some embodiments, the binding protein comprises a second polypeptidechain further comprising a first Fc region linked to CH1, the first Fcregion comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, and a third polypeptidechain further comprising a second Fc region linked to CH1, the second Fcregion comprising an immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains, wherein the first and/orsecond Fc regions comprise amino acid substitutions at positionscorresponding to positions 428 and 434 of human IgG1 or IgG4 accordingto EU Index, wherein the amino acid substitutions are M428L and N434S.In some embodiments, a binding protein of the present disclosurecomprises knob and hole mutations and one or more mutations to improveserum half-life. In some embodiments, the first and/or second Fc regionsare human IgG1 Fc regions. In some embodiments, the first and/or secondFc regions are human IgG4 Fc regions.

In some embodiments, a binding protein of the present disclosurecomprises one or more mutations to reduce effector function, e.g., Fcreceptor-mediated antibody-dependent cellular phagocytosis (ADCP),complement-dependent cytotoxicity (CDC), and/or antibody-dependentcellular cytotoxicity (ADCC). In some embodiments, the secondpolypeptide chain further comprises a first Fe region linked to C_(H1),the first Fc region comprising an immunoglobulin hinge region and C_(H2)and C_(H3) immunoglobulin heavy chain constant domains; wherein thethird polypeptide chain further comprises a second Fc region linked toC_(H1), the second Fc region comprising an immunoglobulin hinge regionand C_(H2) and C_(H3) immunoglobulin heavy chain constant domains;wherein the first and second Fc regions are human IgG1 Fc regions; andwherein the first and the second Fc regions each comprise amino acidsubstitutions at positions corresponding to positions 234 and 235 ofhuman IgG1 according to EU Index, wherein the amino acid substitutionsare L234A and L235A. In some embodiments, the Fc regions of the secondand the third polypeptide chains are human IgG1 Fc regions, and whereinthe Fc regions each comprise amino acid substitutions at positionscorresponding to positions 234 and 235 of human IgG1 according to EUIndex, wherein the amino acid substitutions are L234A and L235A. In someembodiments, the second polypeptide chain further comprises a first Fcregion linked to C_(H1), the first Fc region comprising animmunoglobulin hinge region and C_(H2) and C_(H3) immunoglobulin heavychain constant domains; wherein the third polypeptide chain furthercomprises a second Fc region linked to C_(H1), the second Fc regioncomprising an immunoglobulin hinge region and C_(H2) and C_(H3)immunoglobulin heavy chain constant domains; wherein the first andsecond Fc regions are human IgG1 Fc regions; and wherein the first andthe second Fc regions each comprise amino acid substitutions atpositions corresponding to positions 234, 235, and 329 of human IgG1according to EU Index, wherein the amino acid substitutions are L234A,L235A, and P329A. In some embodiments, the Fc regions of the second andthe third polypeptide chains are human IgG1 Fc regions, and wherein theFc regions each comprise amino acid substitutions at positionscorresponding to positions 234, 235, and 329 of human IgG1 according toEU Index, wherein the amino acid substitutions are L234A, L235A, andP329A. In some embodiments, the Fc regions of the second and the thirdpolypeptide chains are human IgG4 Fc regions, and the Fc regions eachcomprise amino acid substitutions at positions corresponding topositions 234 and 235 of human IgG4 according to EU Index, wherein theamino acid substitutions are F234A and L235A. In some embodiments, thebinding protein comprises a second polypeptide chain further comprisinga first Fc region linked to C_(H1), the first Fc region comprising animmunoglobulin hinge region and C_(H2) and C_(H3) immunoglobulin heavychain constant domains, and a third polypeptide chain further comprisinga second Fc region linked to C_(H1), the second Fc region comprising animmunoglobulin hinge region and C_(H2) and C_(H3) immunoglobulin heavychain constant domains; and wherein the first and the second Fc regionseach comprise amino acid substitutions at positions corresponding topositions 234 and 235 of human IgG4 according to EU Index, wherein theamino acid substitutions are F234A and L235A.

In some embodiments, a binding protein of the present disclosurecomprises knob and hole mutations and one or more mutations to reduceeffector function. In some embodiments, the first and/or second Feregions are human IgG1 Fc regions. In some embodiments, the first and/orsecond Fc regions are human IgG4 Fc regions. For further description ofFc mutations at position 329, see. e.g., Shields, R. L. et al. (2001) J.Biol. Chem. 276:6591-6604 and WO1999051642.

In some embodiments, the types of mutations described supra can becombined in any order or combination. For example, a binding protein ofthe present disclosure can comprise two or more of the “knob” and “hole”mutations, the one or more mutations to improve serum half-life, the oneor more mutations to improve IgG4 stability, the one or more mutationsto improve purification, and/or the one or more mutations to reduceeffector function described supra.

In some embodiments, a binding protein of the present disclosurecomprises an antibody fragment, including but not limited to antibodyF(ab), F(ab′)2, Fab′-SH, Fv, or scFv fragments.

Assays

The present disclosure provides antigen binding proteins that bind humanand/or cynomolgus CD38 polypeptides, induce T cell (e.g., CD4+ and/orCD8+ T cell) proliferation, and/or induce apoptosis of CD38+ cells.Exemplary assays for measuring these parameters and identifying suchbinding proteins are provided herein. For example, in some embodiments,binding affinity between a binding protein or antigen-binding fragmentthereof and a purified CD38 polypeptide is measured by SPR (e.g., asdescribed infra), and binding affinity between a binding protein orantigen-binding fragment thereof and a CD38 polypeptide expressed on thesurface of a cell is measured by flow cytometry (e.g., as describedinfra).

In some embodiments, the antigen binding site of a binding protein ofthe present disclosure binds the human CD38 polypeptide comprising theamino acid sequence of SEQ ID NO:1 with an equilibrium dissociationconstant (K_(D)) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nMor less, 5 nM or less, 2 nM or less, 1 nM or less, or 0.8 nM or less, asmeasured by a flow cytometry assay using cells that express the humanCD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1 ontheir cell surface, e.g., as described infra. In some embodiments, theantigen binding site binds the cynomolgus monkey CD38 polypeptidecomprising the amino acid sequence of SEQ ID NO:30 with an equilibriumdissociation constant (K_(D)) of 20 nM or less, 15 nM or less, 12 nM orless, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, or 0.75nM or less, as measured by a flow cytometry assay using cells thatexpress the cynomolgus monkey CD38 polypeptide comprising the amino acidsequence of SEQ ID NO:30 on their cell surface, e.g., as describedinfra. In some embodiments, the antigen binding site binds the humanCD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1 withan equilibrium dissociation constant (K_(D)) of 20 nM or less, 15 nM orless, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM orless, or 0.83 nM or less, as measured by an SPR assay using the humanCD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1,e.g., as described infra. In some embodiments, the antigen binding sitebinds the cynomolgus monkey CD38 polypeptide comprising the amino acidsequence of SEQ ID NO:30 with an equilibrium dissociation constant(K_(D)) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5nM or less, 3.5 nM or less, 1.5 nM or less, or 1.0 nM or less, asmeasured by an SPR assay using the cynomolgus monkey CD38 polypeptidecomprising the amino acid sequence of SEQ ID NO:30, e.g., as describedinfra. As demonstrated herein, in some embodiments, a binding protein ofthe present disclosure may possess one or more of the exemplary bindingproperties described herein. In some embodiments, the KD is measured at4° C. or 25° C.

In some embodiments, a monospecific binding protein of the presentdisclosure possesses one or more of the following features: binds to theextracellular domain of a human CD38 polypeptide (e.g., comprising theamino acid sequence of SEQ ID NO:1) as a purified protein, as assayed bySPR or ELISA; binds to the extracellular domain of a human CD38polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) asa purified protein with a K_(D) of 20 nM or less, 15 nM or less, 12 nMor less, 10 nM or less, 5 nM or less, 2 nM or less, or 1.5 nM or less,as assayed by SPR or ELISA; binds to the extracellular domain of a humanCD38 polypeptide (e.g., comprising the amino acid sequence of SEQ IDNO:1) expressed on the surface of a cell, as assayed by flow cytometry;binds to the extracellular domain of a human CD38 polypeptide (e.g.,comprising the amino acid sequence of SEQ ID NO:1) expressed on thesurface of a cell with an apparent K_(D) of 20 nM or less, 15 nM orless, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nMor less, as assayed by flow cytometry; binds to the extracellular domainof a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acidsequence of SEQ ID NO:30) as a purified protein, as assayed by SPR orELISA; binds to the extracellular domain of a cynomolgus monkey CD38polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30)as a purified protein with a K_(D) of 20 nM or less, 15 nM or less, 12nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, asassayed by SPR or ELISA; binds to the extracellular domain of acynomolgus monkey CD38 polypeptide (e.g., comprising the amino acidsequence of SEQ ID NO:30) expressed on the surface of a cell, as assayedby flow cytometry; binds to the extracellular domain of a cynomolgusmonkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQID NO:30) expressed on the surface of a cell with an apparent K_(D) of20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM orless, 2 nM or less, 1 nM or less, as assayed by flow cytometry; binds tothe extracellular domain of a human isoform E CD38 polypeptide (e.g.,comprising the amino acid sequence of SEQ ID NO:105) as a purifiedprotein, as assayed by SPR or ELISA; binds to the extracellular domainof a human isoform E CD38 polypeptide (e.g., comprising the amino acidsequence of SEQ ID NO:105) expressed on the surface of a cell, asassayed by flow cytometry; induces apoptosis or antibody-dependentcellular cytotoxicity (ADCC) of a cell expressing CD38 on its cellsurface; and has one or more mutations (e.g., in an Fc region) resultingin decreased binding to FcγRI and/or FcγRIII, as compared to the samebinding protein without the one or more mutations. In some embodiments,a binding protein of the present disclosure binds a CD38 polypeptide(e.g., human or cynomolgus monkey) expressed on the surface of a cellwith an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM orless, 5 nM or less, 2 nM or less, or 1 nM or less, as assayed by flowcytometry. In some embodiments, a binding protein of the presentdisclosure binds a CD38 polypeptide (e.g., human or cynomolgus monkey)as a purified protein with an EC50 of 20 nM or less, 15 nM or less, 12nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less,as assayed by ELISA. In some embodiments, the KD is measured at 4° C. or25° C.

In some embodiments, a trispecific binding protein of the presentdisclosure possesses one or more of the following features: induces Tcell (e.g., CD4+ and/or CD8+ T cell) proliferation; induces T cell(e.g., CD4+ and/or CD8+ T cell) expression of Bcl-xL; induces apoptosisof CD38+ cells (e.g., as measured by Annexin V staining and/or propidiumiodide uptake); binds to CD38 expressed on the surface of a cell and oneor more T cell target antigen(s) expressed on the surface of a T cell;binds to CD38 expressed on the surface of a cell, CD28 expressed on thesurface of a T cell, and CD3 expressed on the surface of a T cell;stimulates activation of the T cell receptor (e.g., as measured by CD69expression); induces costimulation of T cell receptor signaling (e.g.,as mediated by CD28); has one or more mutations (e.g., in an Fc region)resulting in decreased induction of cytokine release (e.g., IFN-γ, IL-2,and/or TNF-α) by PBMCs, as compared to the same binding protein withoutthe one or more mutations; induces cytokine release (e.g., IFN-γ and/orIL-6) by PBMCs in the presence of a CD38+ target cell (e.g., as measuredby immunoassay); binds to the extracellular domain of a human CD38polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) asa purified protein, as assayed by SPR or ELISA; binds to theextracellular domain of a human CD38 polypeptide (e.g., comprising theamino acid sequence of SEQ ID NO:1) as a purified protein with a K_(D)of 1.5 nM or less, as assayed by SPR or ELISA; binds to theextracellular domain of a human CD38 polypeptide (e.g., comprising theamino acid sequence of SEQ ID NO:1) expressed on the surface of a cell,as assayed by flow cytometry; binds to the extracellular domain of ahuman CD38 polypeptide (e.g., comprising the amino acid sequence of SEQID NO:1) expressed on the surface of a cell with an apparent K_(D) of 12nM or less, as assayed by flow cytometry; binds to the extracellulardomain of a cynomolgus monkey CD38 polypeptide (e.g., comprising theamino acid sequence of SEQ ID NO:30) as a purified protein, as assayedby SPR or ELISA; binds to the extracellular domain of a cynomolgusmonkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQID NO:30) as a purified protein with a K_(D) of 3.5 nM or less, asassayed by SPR or ELISA; binds to the extracellular domain of acynomolgus monkey CD38 polypeptide (e.g., comprising the amino acidsequence of SEQ ID NO:30) expressed on the surface of a cell, as assayedby flow cytometry; binds to the extracellular domain of a cynomolgusmonkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQID NO:30) expressed on the surface of a cell with an apparent K_(D) of7.5 nM or less, as assayed by flow cytometry; binds to the extracellulardomain of a human isoform E CD38 polypeptide (e.g., comprising the aminoacid sequence of SEQ ID NO:105) as a purified protein, as assayed by SPRor ELISA; binds to the extracellular domain of a human isoform E CD38polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:105)expressed on the surface of a cell, as assayed by flow cytometry;induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) ofa cell expressing CD38 on its cell surface; and has one or moremutations (e.g., in an Fc region) resulting in decreased binding toFcγRI and/or FcγRII, as compared to the same binding protein without theone or more mutations. In some embodiments, a binding protein of thepresent disclosure binds a CD38 polypeptide (e.g., human or cynomolgusmonkey) expressed on the surface of a cell with an EC50 of 20 nM orless, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM orless, or 1 nM or less, as assayed by flow cytometry. In someembodiments, a binding protein of the present disclosure binds a CD38polypeptide (e.g., human or cynomolgus monkey) as a purified proteinwith an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM orless, 5 nM or less, 2 nM or less, or 1 nM or less, as assayed by ELISA.

Nucleic Acids

Standard recombinant DNA methodologies are used to construct thepolynucleotides that encode the polypeptides which form the bindingproteins, incorporate these polynucleotides into recombinant expressionvectors, and introduce such vectors into host cells. See e.g., Sambrooket al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring HarborLaboratory Press, 3rd ed.). Enzymatic reactions and purificationtechniques may be performed according to manufacturer's specifications,as commonly accomplished in the art, or as described herein. Unlessspecific definitions are provided, the nomenclature utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art. Similarly, conventional techniques may be usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, delivery, and treatment of patients.

Other aspects of the present disclosure relate to isolated nucleic acidmolecules comprising a nucleotide sequence encoding any of the bindingproteins described herein. In some embodiments, the isolated nucleicacid molecules comprise a sequence that is at least 85%, at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to SEQ IDNOs:60-83 and/or shown in Table J.

Certain aspects of the present disclosure relate to kits ofpolynucleotides. In some embodiments, one or more of the polynucleotidesis a vector (e.g., an expression vector). The kits may find use, interalia, in producing one or more of the binding proteins described herein,e.g., a trispecific binding protein of the present disclosure. In someembodiments, the kit comprises one, two, three, or four polynucleotidesshown in Table J (e.g., of mAb2xCD28supxCD3mid IgG4 FALA,mAb2xCD28supxCD3mid IgG1LALA P329A, mAb2xCD28supxCD3mid IgG1 NNSA,mAb6xCD28supxCD3mid IgG4 FALA, mAb6xCD28supxCD3mid IgG1LALA P329A, ormAb6xCD28supxCD3mid IgG1 NNSA). In some embodiments, a kit ofpolynucleotides comprises: a first polynucleotide comprising thesequence of SEQ ID NO:73, a second polynucleotide comprising thesequence of SEQ ID NO:72, a third polynucleotide comprising the sequenceof SEQ ID NO:74, and a fourth polynucleotide comprising the sequence ofSEQ ID NO:75. In some embodiments, a kit of polynucleotides comprises: afirst polynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:76, a thirdpolynucleotide comprising the sequence of SEQ ID NO:77, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:75. In someembodiments, a kit of polynucleotides comprises: a first polynucleotidecomprising the sequence of SEQ ID NO:73, a second polynucleotidecomprising the sequence of SEQ ID NO:78, a third polynucleotidecomprising the sequence of SEQ ID NO:79, and a fourth polynucleotidecomprising the sequence of SEQ ID NO:75. In some embodiments, a kit ofpolynucleotides comprises: a first polynucleotide comprising thesequence of SEQ ID NO:73, a second polynucleotide comprising thesequence of SEQ ID NO:72, a third polynucleotide comprising the sequenceof SEQ ID NO:80, and a fourth polynucleotide comprising the sequence ofSEQ ID NO:81. In some embodiments, a kit of polynucleotides comprises: afirst polynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:76, a thirdpolynucleotide comprising the sequence of SEQ ID NO:82, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:81. In someembodiments, a kit of polynucleotides comprises: a first polynucleotidecomprising the sequence of SEQ ID NO:73, a second polynucleotidecomprising the sequence of SEQ ID NO:78, a third polynucleotidecomprising the sequence of SEQ ID NO:83, and a fourth polynucleotidecomprising the sequence of SEQ ID NO:81.

In some embodiments, the isolated nucleic acid is operably linked to aheterologous promoter to direct transcription of the bindingprotein-coding nucleic acid sequence. A promoter may refer to nucleicacid control sequences which direct transcription of a nucleic acid. Afirst nucleic acid sequence is operably linked to a second nucleic acidsequence when the first nucleic acid sequence is placed in a functionalrelationship with the second nucleic acid sequence. For instance, apromoter is operably linked to a coding sequence of a binding protein ifthe promoter affects the transcription or expression of the codingsequence. Examples of promoters may include, but are not limited to,promoters obtained from the genomes of viruses (such as polyoma virus,fowlpox virus, adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus, Simian Virus 40 (SV40), and the like), from heterologouseukaryotic promoters (such as the actin promoter, an immunoglobulinpromoter, from heat-shock promoters, and the like), the CAG-promoter(Niwa et al., Gene 108(2):193-9, 1991), the phosphoglycerate kinase(PGK)-promoter, a tetracycline-inducible promoter (Masui et al., NucleicAcids Res. 33:e43, 2005), the lac system, the trp system, the tacsystem, the trc system, major operator and promoter regions of phagelambda, the promoter for 3-phosphoglycerate kinase, the promoters ofyeast acid phosphatase, and the promoter of the yeast alpha-matingfactors. Polynucleotides encoding binding proteins of the presentdisclosure may be under the control of a constitutive promoter, aninducible promoter, or any other suitable promoter described herein orother suitable promoter that will be readily recognized by one skilledin the art.

In some embodiments, the isolated nucleic acid is incorporated into avector. In some embodiments, the vector is an expression vector.Expression vectors may include one or more regulatory sequencesoperatively linked to the polynucleotide to be expressed. The term“regulatory sequence” includes promoters, enhancers and other expressioncontrol elements (e.g., polyadenylation signals). Examples of suitableenhancers may include, but are not limited to, enhancer sequences frommammalian genes (such as globin, elastase, albumin, α-fetoprotein,insulin and the like), and enhancer sequences from a eukaryotic cellvirus (such as SV40 enhancer on the late side of the replication origin(bp 100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, adenovirusenhancers, and the like). Examples of suitable vectors may include, forexample, plasmids, cosmids, episomes, transposons, and viral vectors(e.g., adenoviral, vaccinia viral, Sindbis-viral, measles, herpes viral,lentiviral, retroviral, adeno-associated viral vectors, etc.).Expression vectors can be used to transfect host cells, such as, forexample, bacterial cells, yeast cells, insect cells, and mammaliancells. Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art, and can beused to transfect any cell of interest.

Other aspects of the present disclosure relate to a vector systemcomprising one or more vectors encoding a first, second, third, andfourth polypeptide chain of any of the binding proteins describedherein. In some embodiments, the vector system comprises a first vectorencoding the first polypeptide chain of the binding protein, a secondvector encoding the second polypeptide chain of the binding protein, athird vector encoding the third polypeptide chain of the bindingprotein, and a fourth vector encoding the fourth polypeptide chain ofthe binding protein. In some embodiments, the vector system comprises afirst vector encoding the first and second polypeptide chains of thebinding protein, and a second vector encoding the third and fourthpolypeptide chains of the binding protein. In some embodiments, thevector system comprises a first vector encoding the first and thirdpolypeptide chains of the binding protein, and a second vector encodingthe second and fourth polypeptide chains of the binding protein. In someembodiments, the vector system comprises a first vector encoding thefirst and fourth polypeptide chains of the binding protein, and a secondvector encoding the second and third polypeptide chains of the bindingprotein. In some embodiments, the vector system comprises a first vectorencoding the first, second, third, and fourth polypeptide chains of thebinding protein. The one or more vectors of the vector system may be anyof the vectors described herein. In some embodiments, the one or morevectors are expression vectors.

Isolated Host Cells

Other aspects of the present disclosure relate to an isolated host cellcomprising one or more isolated polynucleotides, polynucleotide kits,vectors, and/or vector systems described herein. In some embodiments,the host cell is a bacterial cell (e.g., an E. coli cell). In someembodiments, the host cell is a yeast cell (e.g. an S. cerevisiae cell).In some embodiments, the host cell is an insect cell. Examples of insecthost cells may include, for example, Drosophila cells (e.g., S2 cells),Trichoplusia ni cells (e.g., High Five™ cells), and Spodopterafrugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, thehost cell is a mammalian cell. Examples of mammalian host cells mayinclude, for example, human embryonic kidney cells (e.g., 293 or 293cells subcloned for growth in suspension culture), Expi293TM cells, CHOcells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse sertolicells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70),African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), humancervical carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells(e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCCCRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells(e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562,ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, a human hepatoma line(e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2/0 cells).

Other aspects of the present disclosure relate to a method of producingany of the binding proteins described herein. In some embodiments, themethod includes a) culturing a host cell (e.g., any of the host cellsdescribed herein) comprising an isolated nucleic acid, vector, and/orvector system (e.g., any of the isolated nucleic acids, vectors, and/orvector systems described herein) under conditions such that the hostcell expresses the binding protein; and b) isolating the binding proteinfrom the host cell. Methods of culturing host cells under conditions toexpress a protein are well known to one of ordinary skill in the art.Methods of isolating proteins from cultured host cells are well known toone of ordinary skill in the art, including, for example, by affinitychromatography (e.g., two step affinity chromatography comprisingprotein A affinity chromatography followed by size exclusionchromatography).

In some embodiments, a binding protein of the present disclosure ispurified by protein A affinity chromatography, kappa light chainaffinity chromatography (e.g., using a KappaSelect resin according tomanufacturer's instructions; GE Healthcare), and optionally lambda lightchain affinity chromatography (e.g., using a LambdaFabSelect resinaccording to manufacturer's instructions; GE Healthcare). In someembodiments, a binding protein of the present disclosure is purified byProtein A affinity chromatography, lambda light chain affinitychromatography (e.g., using a LambdaFabSelect resin according tomanufacturer's instructions; GE Healthcare), and optionally kappa lightchain affinity chromatography (e.g., using a KappaSelect resin accordingto manufacturer's instructions; GE Healthcare). In some embodiments, thebinding protein comprises two Fc regions, each comprising a C_(H3)domain, and only one of the C_(H3) domains comprises amino acidsubstitutions at positions corresponding to positions 435 and 436 ofhuman IgG1 or IgG4 according to EU Index, wherein the amino acidsubstitutions are H435R and Y436F. In some embodiments, a bindingprotein of the present disclosure is purified by protein A affinitychromatography, then kappa light chain affinity chromatography (e.g.,using a KappaSelect resin according to manufacturer's instructions; GEHealthcare), then optionally lambda light chain affinity chromatography(e.g., using a LambdaFabSelect resin according to manufacturer'sinstructions; GE Healthcare) in sequence. In some embodiments, a bindingprotein of the present disclosure is purified by Protein A affinitychromatography, then lambda light chain affinity chromatography (e.g.,using a LambdaFabSelect resin according to manufacturer's instructions;GE Healthcare), then optionally kappa light chain affinitychromatography (e.g., using a KappaSelect resin according tomanufacturer's instructions; GE Healthcare) in sequence. For example, insome embodiments, the binding protein is contacted with Protein A,eluted from Protein A under conditions suitable for isolating thebinding protein away from binding proteins comprising either 0 or 2C_(H3) domains comprising the amino acid substitutions are H435R andY436F, contacted with a kappa light chain affinity medium (e.g., as usedin the KappaSelect resin; GE Healthcare), and eluted from the kappalight chain affinity medium under conditions suitable for isolating thebinding protein away from binding proteins comprising only lambda C_(L)domains (e.g., according to manufacturer's instructions). Conditionssuitable for the Protein A elution are known in the art, includingwithout limitation a stepwise elution gradient from pH4.5-2.8. In someembodiments, Protein A or a Protein A variant useful for proteinpurification is employed. In some embodiments, the Protein A is attachedto a substrate or resin, e.g., as part of a chromatography medium. Insome embodiments, after elution from the kappa light chain affinitymedium, the binding protein is contacted with a lambda light chainaffinity medium (e.g., as used in the LambdaFabSelect resin; GEHealthcare), and eluted from the lambda light chain affinity mediumunder conditions suitable for isolating the binding protein away frombinding proteins comprising only kappa C_(L) domains (e.g., according tomanufacturer's instructions). In some embodiments, a binding protein ofthe present disclosure is detected using HIC chromatography. In someembodiments, the binding protein comprises: a first polypeptide chainthat comprises a lambda C_(L) domain; a C_(H3) domain of a secondpolypeptide chain that comprises amino acid substitutions at positionscorresponding to positions 354 and 366 of human IgG1 or IgG4 accordingto EU Index, wherein the amino acid substitutions are S354C and T366W; aC_(H3) domain of a third polypeptide chain that comprises amino acidsubstitutions at positions corresponding to positions 349, 366, 368,407, 435, and 436 of human IgG1 or IgG4 according to EU Index, whereinthe amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, andY436F; and a fourth polypeptide chain that comprises a kappa C_(L)domain. In some embodiments, the binding protein is produced by a hostcell. In some embodiments, the binding protein is purified from a cellculture medium or host cell extract. In some embodiments, the bindingproteins are secreted by a host cell or produced and extracted from ahost cell (e.g., before being contacted with Protein A). In someembodiments, the binding protein is in a cell culture medium or hostcell extract when contacted with Protein A. In some embodiments, thebinding protein is purified away from other binding proteins,polypeptides, and/or other cellular components.

III. Trispecific Binding Proteins

In some embodiments, the binding protein of the disclosure is atrispecific and/or trivalent binding protein comprising four polypeptidechains that form three antigen binding sites that bind one or more(e.g., three) different antigen targets or target proteins. In someembodiments, a first polypeptide chain comprises a structure representedby the formula:

V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I]

and a second polypeptide chain comprises a structure represented by theformula:

V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3)  [II]

and a third polypeptide chain comprises a structure represented by theformula:

V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3)  [III]

and a fourth polypeptide chain comprises a structure represented by theformula:

V_(L3)-C_(L)  [IV]

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(L3) is a third immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(L2) is a second immunoglobulin heavy chain variable domain;

V_(H3) is a third immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H1) is an immunoglobulin C_(H1) heavy chain constant domain;

C_(H2) is an immunoglobulin C_(H2) heavy chain constant domain;

C_(H3) is an immunoglobulin C_(H3) heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the C_(H1) and C_(H2)domains; and

L₁, L₂, L₃ and L₄ are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula IIform a cross-over light chain-heavy chain pair. In some embodiments, thefirst polypeptide chain and the second polypeptide chain have across-over orientation that forms two distinct antigen binding sites. Asdescribed above, the second and third polypeptide chains include an Fcregion (e.g., comprising the hinge-C_(H2)-C_(H3) domains). In someembodiments, one or both Fc regions are human IgG4 Fc regions comprisingamino acid substitutions at positions corresponding to positions 233-236of human IgG4 according to EU Index. In some embodiments, the amino acidsubstitutions are E233P, F234V, L235A, and a deletion at 236. In someembodiments, the Fc regions are human IgG4 Fc regions comprising aminoacid mutations at substitutions corresponding to positions 228, 233-236,and/or 409 of human IgG4 according to EU Index. In some embodiments, theamino acid mutations are S228P; E233P, F234V, L235A, and a deletion at236; and/or R409K. In some embodiments, one or both Fc regions are humanIgG1 Fc regions comprising one or more amino acid substitutions atpositions corresponding to positions 234, 235, and/or 329 of human IgG1according to EU Index. In some embodiments, the amino acid substitutionsare L234A, L235A, and/or P329A. In some embodiments, the Fc regions arehuman IgG1 Fc regions comprising amino acid substitutions at positionscorresponding to positions 298, 299, and/or 300 of human IgG1 accordingto EU Index. In some embodiments, the amino acid substitutions areS298N, T299A, and/or Y300S.

In some embodiments, the binding protein of the disclosure is atrispecific and/or trivalent binding protein comprising four polypeptidechains that form three antigen binding sites that bind one or more(e.g., three) different antigen targets or target proteins. In someembodiments, at least one of the antigen binding sites binds a CD38polypeptide (e.g., the extracellular domain of human and/or cynomolgusmonkey CD38 polypeptides). In some embodiments, a first polypeptidechain comprises a structure represented by the formula:

V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I]

and a second polypeptide chain comprises a structure represented by theformula:

V_(H1)-L₃-V_(H2)-L₄-C_(H1)  [II]

and a third polypeptide chain comprises a structure represented by theformula:

V_(H3)-C_(H1)  [III]

and a fourth polypeptide chain comprises a structure represented by theformula:

V_(L3)-C_(L)  [IV]

wherein:

V_(L1) is a first immunoglobulin light chain variable domain;

V_(L2) is a second immunoglobulin light chain variable domain;

V_(L3) is a third immunoglobulin light chain variable domain;

V_(H1) is a first immunoglobulin heavy chain variable domain;

V_(H2) is a second immunoglobulin heavy chain variable domain;

V_(H3) is a third immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H1) is an immunoglobulin C_(H1) heavy chain constant domain; and

L₁, L₂, L₃ and L₄ are amino acid linkers;

and wherein the polypeptide of formula I and the polypeptide of formulaII form a cross-over light chain-heavy chain pair. In some embodiments,the second and the third polypeptide chain further comprise an Fc regionlinked to C_(H1), the Fc regions comprising an immunoglobulin hingeregion and C_(H2) and C_(H3) immunoglobulin heavy chain constantdomains. In some embodiments, one or both Fc regions are human IgG4 Fcregions comprising amino acid substitutions at positions correspondingto positions 233-236 of human IgG4 according to EU Index. In someembodiments, the amino acid substitutions are E233P, F234V, L235A, and adeletion at 236. In some embodiments, the Fc regions are human IgG4 Fcregions comprising amino acid mutations at substitutions correspondingto positions 228, 233-236, and/or 409 of human IgG4 according to EUIndex. In some embodiments, the amino acid mutations are S228P; E233P,F234V, L235A, and a deletion at 236; and/or R409K. In some embodiments,one or both Fc regions are human IgG1 Fc regions comprising one or moreamino acid substitutions at positions corresponding to positions 234,235, and/or 329 of human IgG1 according to EU Index. In someembodiments, the amino acid substitutions are L234A, L235A, and/orP329A. In some embodiments, the Fc regions are human IgG1 Fc regionscomprising amino acid substitutions at positions corresponding topositions 298, 299, and/or 300 of human IgG1 according to EU Index. Insome embodiments, the amino acid substitutions are S298N, T299A, and/orY300S.

In some embodiments, the VH1 and VL1 form a binding pair and form afirst antigen binding site. In some embodiments, the VH2 and VL2 form abinding pair and form a second antigen binding site. In someembodiments, the VH3 and VL3 form a binding pair and form a thirdantigen binding site. The binding proteins can also be used for cellactivation, tumor targeting, neutralization of cytokine activities,neutralization of viral infection, combination of multiple signalingevents, to treat cancer, arthritis, and/or inflammatory disorders. Forexample, in some embodiments, a binding protein specifically binds one,two, or three antigen targets selected from A2AR, APRIL, ATPDase, BAFF,BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B71-1, B7H4 (also known as VTCN1),B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3(also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known asRANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11(also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also knownas TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a),CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2),CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4,CD3, CD19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24,CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86(also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152(also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273(also known as PDL2), CD274 (also known as PDL1), CD275 (also known asB7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279(also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9,CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known asGM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1),CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleosidetriphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP,FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO,IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra,IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13,IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor forIL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1,NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152,SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Sykkinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1,TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor forIL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known asGPR5/CCXCR1). In some embodiments, one or more of the above antigentargets are human antigen targets.

In some embodiments, one of the three antigen binding sites binds a CD3polypeptide (e.g., a human CD3 polypeptide), one of the three antigenbinding sites binds a CD28 polypeptide (e.g., a human CD28 polypeptide),and one of the three antigen binding sites binds a third polypeptide. Insome embodiments, the antigen binding site that specifically binds anantigen target other than CD3 or CD28 binds an antigen target selectedfrom A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC,B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5,CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (alsoknown as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known asMCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15(also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known asMIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2),CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK),CCL26 (also known as eotaxin-3), CCR3, CCR4, CD19, CD20, CD23 (alsoknown as FCER2, a receptor for IgE), CD24, CD27, CD38, CD39, CD40, CD70,CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (alsoknown as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also knownas CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also knownas PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278(also known as ICOS), CD279 (also known as PD-1), CDH1 (also known asE-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known asM-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1(also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3,DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1,FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2,HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B,IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R,IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (alsoknown as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35,ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2,MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, plateletreceptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptorfor IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2,TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known asa co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, andXCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more ofthe above antigen targets are human antigen targets.

In any of the trispecific binding proteins described supra, any linkeror combination of linkers described herein may be used. For example, insome embodiments, at least one of L₁, L₂, L₃ or L₄ is independently 0amino acids in length. In some embodiments, L₁, L₂, L₃ or L₄ are eachindependently at least one amino acid in length. In some embodiments,L₁, L₂, L₃ and L₄ each independently are zero amino acids in length orcomprise a sequence selected from the group consisting of GGGGSGGGGS(SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ IDNO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59). In someembodiments, L₁, L₂, L₃ and L₄ each independently comprise a sequenceselected from the group consisting of GGGGSGGGGS (SEQ ID NO:55),GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP(SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L₁comprises the sequence GQPKAAP (SEQ ID NO: 58), L₂ comprises thesequence TKGPS (SEQ ID NO:57), L₃ comprises the sequence S, and L₄comprises the sequence RT. In some embodiments, L₁ comprises thesequence GGGGSGGGGS (SEQ ID NO:55), L₂ comprises the sequence GGGGSGGGGS(SEQ ID NO:55), L₃ is 0 amino acids in length, and L₄ is 0 amino acidsin length. In some embodiments, L₁ comprises the sequence GGSGSSGSGG(SEQ ID NO:59), L₂ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₃is 0 amino acids in length, and L₄ is 0 amino acids in length. In someembodiments, L₁ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56),L₂ is 0 amino acids in length, L₃ comprises the sequence GGGGSGGGGSGGGGS(SEQ ID NO:56), and L₄ is 0 amino acids in length.

IV. Uses for Binding Proteins

The binding proteins can be employed in any known assay method, such ascompetitive binding assays, direct and indirect sandwich assays, andimmunoprecipitation assays for the detection and quantitation of one ormore target antigens. The binding proteins will bind the one or moretarget antigens with an affinity that is appropriate for the assaymethod being employed.

For diagnostic applications, in certain embodiments, binding proteinscan be labeled with a detectable moiety. The detectable moiety can beany one that is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety can be aradioisotope, such as ³H, ⁴C, ³²P, ³⁵S, ¹²⁵I, ⁹⁹Tc, ¹¹¹In, or ⁶⁷Ga; afluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, β-galactosidase, or horseradish peroxidase.

The binding proteins are also useful for in vivo imaging. A bindingprotein labeled with a detectable moiety can be administered to ananimal, preferably into the bloodstream, and the presence and locationof the labeled antibody in the host assayed. The binding protein can belabeled with any moiety that is detectable in an animal, whether bynuclear magnetic resonance, radiology, or other detection means known inthe art.

For clinical or research applications, in certain embodiments, bindingproteins can be conjugated to a cytotoxic agent. A variety of antibodiescoupled to cytotoxic agents (i.e., antibody-drug conjugates) have beenused to target cytotoxic payloads to specific tumor cells. Cytotoxicagents and linkers that conjugate the agents to an antibody are known inthe art; see, e.g., Parslow, A. C. et al. (2016) Biomedicines 4:14 andKalim, M. et al. (2017) Drug Des. Devel. Ther. 11:2265-2276.

The disclosure also relates to a kit comprising a binding protein andother reagents useful for detecting target antigen levels in biologicalsamples. Such reagents can include a detectable label, blocking serum,positive and negative control samples, and detection reagents. In someembodiments, the kit comprises a composition comprising any bindingprotein, polynucleotide, vector, vector system, and/or host celldescribed herein. In some embodiments, the kit comprises a container anda label or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, IV solutionbags, etc. The containers may be formed from a variety of materials suchas glass or plastic. The container holds a composition which is byitself or combined with another composition effective for treating,preventing and/or diagnosing a condition and may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Insome embodiments, the label or package insert indicates that thecomposition is used for preventing, diagnosing, and/or treating thecondition of choice. Alternatively, or additionally, the article ofmanufacture or kit may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

In some embodiments, a binding protein of the present disclosure isadministered to a patient in need thereof for the treatment orprevention of cancer. In some embodiments, the present disclosurerelates to a method of preventing and/or treating a proliferativedisease or disorder (e.g., cancer). In some embodiments, the methodcomprises administering to a patient a therapeutically effective amountof at least one of the binding proteins, or pharmaceutical compositionsrelated thereto, described herein. In some embodiments, the presentdisclosure relates to uses of at least one of the binding proteins, orpharmaceutical compositions related thereto, described herein forpreventing and/or treating a proliferative disease or disorder (e.g.,cancer) in a patient in need thereof. In some embodiments, the presentdisclosure relates to at least one of the binding proteins, orpharmaceutical compositions related thereto, described herein for use inthe manufacture of a medicament for preventing and/or treating aproliferative disease or disorder (e.g., cancer) in a patient in needthereof. In some embodiments, the patient is a human. In someembodiments, the binding protein comprises one antigen binding site thatbinds a T-cell surface protein and another antigen binding site thatbinds the extracellular domain of a human CD38 polypeptide, e.g., asdescribed in section II supra. In some embodiments, the binding proteincomprises an antigen binding site that binds the extracellular domain ofa human CD38 polypeptide, an antigen binding site that binds a humanCD28 polypeptide, and an antigen binding site that binds a human CD3polypeptide.

In some embodiments, cells of the cancer express a human CD38 isoform Apolypeptide on their cell surface (e.g., comprising the amino acidsequence of SEQ ID NO:1). In some embodiments, cells of the cancerexpress a human CD38 isoform E polypeptide on their cell surface (e.g.,comprising the amino acid sequence of SEQ ID NO:105). In someembodiments, the patient is selected for treatment on the basis that thecells of the cancer express a human CD38 isoform E polypeptide on theircell surface (e.g., comprising the amino acid sequence of SEQ IDNO:105). In some embodiments, the cancer cells express CD38 and CD28. Insome embodiments, the cancer cells express CD38 and do not express CD28.

In some embodiments, the cancer is multiple myeloma, acute lymphoblasticleukemia, chronic lymphocytic leukemia, acute myeloid leukemia,lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer,germinal center B-cell lympohoma or B-cell acute lymphoblastic leukemia.In certain embodiments, the cancer is multiple myeloma. In certainembodiments, the cancer is acute myeloid leukemia (AML), acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or a Bcell lymphoma.

In certain embodiments, the cancer is multiple myeloma. Anti-CD38antibodies have been tested for the treatment of multiple myeloma, suchas daratumumab and isatuximab. However, while multiple myeloma isconsidered treatable, relapse is inevitable in almost all patients,leading to the development of treatment-refractory disease. In someembodiments, the cancer is relapsed or refractory multiple myeloma. Insome embodiments, the patient has been treated with a prior multiplemyeloma treatment. In some embodiments, a binding protein of the presentdisclosure is administered to the patient as a 1^(st), 2^(nd), or 3^(rd)line treatment for multiple myeloma. Without wishing to be bound totheory, it is thought that an anti-CD38xanti-CD28xanti-CD3 bindingprotein of the present disclosure may be useful in treating multiplemyeloma, e.g., by recruiting T cells to tumor cells via anti-CD38 (oranti-CD28/anti-CD38), activation of engaged T cells viaanti-CD3/anti-CD28, and/or killing of tumor cells throughperforin/granzyme-based mechanisms. CD28 has been reported as a novelcancer marker for multiple myeloma. See Nair, J. R. et al. (2011) J.Immunol. 187:1243-1253.

In some embodiments, the at least one binding protein is administered(or is to be administered) in combination with one or more anti-cancertherapies (e.g., any anti-cancer therapy known in the art, such as achemotherapeutic agent or therapy). In some embodiments, the at leastone binding protein is administered (or is to be administered) beforethe one or more anti-cancer therapies. In some embodiments, the at leastone binding protein is administered (or is to be administered)concurrently with the one or more anti-cancer therapies. In someembodiments, the at least one binding protein is administered (or is tobe administered) after the one or more anti-retroviral therapies.

V. Binding Protein Therapeutic Compositions and Administration Thereof

Therapeutic or pharmaceutical compositions comprising binding proteinsare within the scope of the disclosure. Such therapeutic orpharmaceutical compositions can comprise a therapeutically effectiveamount of a binding protein, or binding protein-drug conjugate, inadmixture with a pharmaceutically or physiologically acceptableformulation agent selected for suitability with the mode ofadministration.

Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed.

The pharmaceutical composition can contain formulation materials formodifying, maintaining, or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. Suitable formulation materials include, but are not limitedto, amino acids (such as glycine, glutamine, asparagine, arginine, orlysine), antimicrobials, antioxidants (such as ascorbic acid, sodiumsulfite, or sodium hydrogen-sulfite), buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids),bulking agents (such as mannitol or glycine), chelating agents (such asethylenediamine tetraacetic acid (EDTA)), complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin, gelatin, orimmunoglobulins), coloring, flavoring and diluting agents, emulsifyingagents, hydrophilic polymers (such as polyvinylpyrrolidone), lowmolecular weight polypeptides, salt-forming counterions (such assodium), preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),solvents (such as glycerin, propylene glycol, or polyethylene glycol),sugar alcohols (such as mannitol or sorbitol), suspending agents,surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;polysorbates such as polysorbate 20 or polysorbate 80; triton;tromethamine; lecithin; cholesterol or tyloxapal), stability enhancingagents (such as sucrose or sorbitol), tonicity enhancing agents (such asalkali metal halides—preferably sodium or potassium chloride—or mannitolsorbitol), delivery vehicles, diluents, excipients and/or pharmaceuticaladjuvants (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editionsof the same, incorporated herein by reference for any purpose).

The optimal pharmaceutical composition will be determined by a skilledartisan depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. Such compositionscan influence the physical state, stability, rate of in vivo release,and rate of in vivo clearance of the binding protein.

The primary vehicle or carrier in a pharmaceutical composition can beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier for injection can be water, physiological saline solution, orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichcan further include sorbitol or a suitable substitute. In one embodimentof the disclosure, binding protein compositions can be prepared forstorage by mixing the selected composition having the desired degree ofpurity with optional formulation agents in the form of a lyophilizedcake or an aqueous solution. Further, the binding protein can beformulated as a lyophilizate using appropriate excipients such assucrose.

The pharmaceutical compositions of the disclosure can be selected forparenteral delivery or subcutaneous. Alternatively, the compositions canbe selected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at a slightly lowerpH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use can be in the form of a pyrogen-free, parenterallyacceptable, aqueous solution comprising the desired binding protein in apharmaceutically acceptable vehicle. A particularly suitable vehicle forparenteral injection is sterile distilled water in which a bindingprotein is formulated as a sterile, isotonic solution, properlypreserved. Yet another preparation can involve the formulation of thedesired molecule with an agent, such as injectable microspheres,bio-erodible particles, polymeric compounds (such as polylactic acid orpolyglycolic acid), beads, or liposomes, that provides for thecontrolled or sustained release of the product which can then bedelivered via a depot injection. Hyaluronic acid can also be used, andthis can have the effect of promoting sustained duration in thecirculation. Other suitable means for the introduction of the desiredmolecule include implantable drug delivery devices.

In one embodiment, a pharmaceutical composition can be formulated forinhalation. For example, a binding protein can be formulated as a drypowder for inhalation. Binding protein inhalation solutions can also beformulated with a propellant for aerosol delivery. In yet anotherembodiment, solutions can be nebulized.

It is also contemplated that certain formulations can be administeredorally. In one embodiment of the disclosure, binding proteins that areadministered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. For example, a capsule can be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the binding protein. Diluents, flavorings,low melting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders can also be employed.

Another pharmaceutical composition can involve an effective quantity ofbinding proteins in a mixture with non-toxic excipients that aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or another appropriate vehicle, solutions can be preparedin unit-dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions of the disclosure will be evidentto those skilled in the art, including formulations involving bindingproteins in sustained- or controlled-delivery formulations. Techniquesfor formulating a variety of other sustained- or controlled-deliverymeans, such as liposome carriers, bio-erodible microparticles or porousbeads and depot injections, are also known to those skilled in the art.Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides, copolymers of L-glutamic acid andgamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylenevinyl acetate, or poly-D(−)-3-hydroxybutyric acid. Sustained-releasecompositions can also include liposomes, which can be prepared by any ofseveral methods known in the art.

Pharmaceutical compositions to be used for in vivo administrationtypically must be sterile. This can be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method can be conducted eitherprior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration can be stored in lyophilizedform or in a solution. In addition, parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it can bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations can bestored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration.

The disclosure also encompasses kits for producing a single-doseadministration unit. The kits can each contain both a first containerhaving a dried protein and a second container having an aqueousformulation. Also included within the scope of this disclosure are kitscontaining single and multi-chambered pre-filled syringes (e.g., liquidsyringes and lyosyringes).

The effective amount of a binding protein pharmaceutical composition tobe employed therapeutically will depend, for example, upon thetherapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich the binding protein is being used, the route of administration,and the size (body weight, body surface, or organ size) and condition(the age and general health) of the patient. Accordingly, the cliniciancan titer the dosage and modify the route of administration to obtainthe optimal therapeutic effect.

Dosing frequency will depend upon the pharmacokinetic parameters of thebinding protein in the formulation being used. Typically, a clinicianwill administer the composition until a dosage is reached that achievesthe desired effect. The composition can therefore be administered as asingle dose, as two or more doses (which may or may not contain the sameamount of the desired molecule) over time, or as a continuous infusionvia an implantation device or catheter. Further refinement of theappropriate dosage is routinely made by those of ordinary skill in theart and is within the ambit of tasks routinely performed by them.Appropriate dosages can be ascertained through use of appropriatedose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally; through injection byintravenous, intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial,intraportal, or intralesional routes; by sustained release systems; orby implantation devices. Where desired, the compositions can beadministered by bolus injection or continuously by infusion, or byimplantation device.

The composition can also be administered locally via implantation of amembrane, sponge, or other appropriate material onto which the desiredmolecule has been absorbed or encapsulated. Where an implantation deviceis used, the device can be implanted into any suitable tissue or organ,and delivery of the desired molecule can be via diffusion, timed-releasebolus, or continuous administration.

EXAMPLES

The Examples that follow are illustrative of specific embodiments of thedisclosure, and various uses thereof. They are set forth for explanatorypurposes only, and should not be construed as limiting the scope of theinvention in any way.

The following terminology may be used interchangeably in the Examplesand Drawings herein to refer to specific anti-CD38 antigen bindingdomains or antibodies:

antiCD38_C2-CD38-1: mAb1

antiCD38_C2-CD38-1_VH1-VL1 or CD38_(VH1): mAb2

antiCD38_C2-CD38-1_VH3-VL3: mAb3

antiCD38_C2-CD38-1_VH5-VL3: mAb4

antiCD38_C2-CD38-1_VH6-VL3: mAb5

antiCD38_1370 or CD38_(HHY1370): mAb6

antiCD38_SB19 or isatuximab: mAb7.

Example 1: Generation and Characterization of Monoclonal Anti-CD38Antibodies

The Examples that follow describe the generation and characterization ofmonoclonal anti-CD38 antibodies. Advantageously, antibodies providedherein cross-react with human and monkey CD38 proteins, therebyproviding molecules that can be used for both safety and clinicalstudies. These antibodies are also capable of killing CD38+ cells byapoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC).

This Example describes an efficient workflow for generatingCD38-specific and cross-reactive monoclonal antibodies from singlemurine B cells.

Materials and Methods

Generation of Monoclonal Antibodies

Antibodies to human CD38 were generated using the human CD38extracellular domain R45-1300 (SEQ ID NO:1). See Q. Liu, I. Krilksunov,R. Graeff, C. Munshi, H. C. Lee, and Q. Hao 2005 Structure 13::1331-1339. The immunogen was administered directly, with an adjuvant tostimulate the immune response, to either normal BalbC mice or transgenicTrianni Mice™ (Trianni, San Francisco, Calif.) comprising DNA encodinghuman Immunoglobulin heavy and kappa light chain variable regions.

Various recombinant CD38 proteins derived from isoform A with differenttag and point mutations were used (SEQ ID NOs:2, 3, 4, and 28), and atagged version of CD38 isoform E (SEQ ID NO: 105) encompassing CD38extracellular domain from R45-P203. The proteins were produced bytransient expression in mammalian cells. Coding DNA sequences werecloned into mammalian expression plasmids under CMV enhancer/promoterand SV40 polyA signals. HEK293 cells (Invitrogen; #K9000-10) weretransiently transfected with the expression plasmids using FreeStyle™MAX 293 Expression System according to the manufacturer's instructions.

Immunization of Mice and Single B Cell Selection

Anti-CD38 antibodies were also isolated directly from antigen-positive Bcells without fusion to myeloma cells. Using this method, severalanti-CD38 antibodies were obtained, such as mAb1 (see SEQ ID Nos: 5 and6 for VH and VL sequences, respectively, and SEQ ID Nos: 7 and 8 forheavy chain and light chain sequences, respectively). Briefly, 6-8 weeksold female BALB/c mice (S082342; Charles River Labs, Bar Harbor, Me.)each received three rounds of immunization over a course of 41 daysusing the classical method as described by A. Wennerberg et al. (1993Am. J. Pathol. 143:1050-1054). Antigen was administeredintraperitoneally to ventral site of mice. Three days after the lastinjection, mice were sacrificed and spleens were isolated asepticallyand washed with fresh RPMI medium. Lymphocytes were released from thespleens and single-cell suspension was washed twice with RPMI mediumbefore being sorted using a four colour sorting strategy including apanel of fluorescent antibodies and dual human and monkey CD38 proteinsand then were separated using flow cytometric cell sorting to isolatehuman/monkey cross reactive IgG-CD38 specific B cells. Single cells weredirectly sorted into PCR tubes to amplify cognate pair of VH and VLgenes by RT-PCR (T. Tiller, C. Busse and H. Wardemann 2009 J. Immunol.Methods 350:183-193). Resulting DNA was sequenced.

Resulting DNA was cloned into a mammalian expression vector encodingrespectively the human IgG1 or human Ck domains for transient expressionin HEK293 cells using FreeStyle™ MAX 293 Expression System according tothe manufacturer's instructions. Batches were purified by protein Aaffinity chromatography (MabSelect, GE Heathcare). The eluate wasdialyzed against PBS before sterile filtration and storage at 4° C.

Generation of Antibodies by Immunization in Human ImmunoglobulinTransgenic Mice and Selection Using Hybridoma Technology

Immunizations, fusion and screening were performed using P3X63-Ag8.653myeloma cells with the extracellular domain of human CD38 as describedin Kilpatrick et al. 1997 Hybridoma 16: 381389. Using the RIMMS methoddescribed by Kilpatrick et al., 6-8 week old female transgenic TrianniMice™ comprising DNA encoding human Immunoglobulin heavy and kappa lightchain variable regions each received four rounds of immunization over acourse of 14 days at intervals of 3-4 days. CD38 protein emulsified inRIBI's adjuvant (Sigma #T2684) was administered subcutaneously to sixsites proximal to draining lymph nodes, along the back of the mice andto six juxtaposed sites along abdomen. Four days after the lastinjection, mice were sacrificed. Bilateral popliteal, superficialinguinal, axillary and branchial lymph nodes were isolated asepticallyand washed with fresh RPMI medium. Lymphocytes were released from thelymph nodes and single-cell suspension was washed twice with RPMI mediumbefore being fused with P3X63-AG8.653 myeloma cells using polyethyleneglycol. After fusion, the cell mixture was incubated in an incubator at37° C. for 16-24 hours. The resulting cell preparation was transferredinto selective semi-solid medium and aseptically plated out into 100 mmPetri plates and incubated at 37° C. Ten days after initiation ofselection, the plates were examined for hybridoma growth, and visiblecolonies were picked-up and placed into 96-well plates containing 200 μLof growth medium. The 96-well plates were kept in an incubator at 37° C.for 2 to 4 days. Using this technique, and the immunogen describedabove, several anti-CD38 chimeric antibodies were obtained such as mAb 6(see SEQ ID Nos: 9 and 10 for VH and VL sequences, respectively, and SEQID Nos: 11 and 12 for heavy chain and light chain sequences,respectively. The VH and VL sequences were retrieved by RT-PCR and mAb 6was produced by transient expression as described above.

Binding Affinity to Soluble CD38 Extracellular Domains

The binding properties of the anti-huCD38 mabs were evaluated using aBIAcore 2000 (BIAcore Inc., Uppsala, N.J.). Briefly, a CM5 BIAcorebiosensor chip was docked into the instrument and activated with 250 μLof 1:1 NHS/EDC at room temperature. A mouse anti-human Fc IgG1 (GEHealthcare #BR-1008-39) (13.5 μg/mL in 0.05M acetate buffer, pH5) wereimmobilized on the activated chips in flow cells 1. The immobilizationwas carried out at a flow rate of 5 μL/min. The chip was then blocked byinjection of 55 μL of ethanolamine-HCl, pH8.5, followed by five washeswith 50 mM NaOH, 1M NaCl. To measure the binding of anti-CD38 mabs tothe human CD38 protein or cyno CD38 protein, antibodies were used at 2μg/mL in BIAcore running buffer (HBS-EP). Antigens (human CD38-histag(ID2) or cyno CD38-histag (ID3)) were injected from 3 to 1000 nM.Following completion of the injection phase, dissociation was monitoredin a BIAcore running buffer at the same flow rate for 360 sec. Thesurface was regenerated between injections using 30 μL of 50 mM NaOH-1 MNaCl. Individual sensorgrams were analyzed using BIAsimulation software.

Binding Affinity to Human CD38-Expressing Pre-B Cells

The binding of anti-CD38 antibodies to CD38 expressed on the surface ofrecombinant murine preB::300.19 cells was determined by flow cytometry.The recombinant cell line was described by J. Deckket et al. 2014 Clin.Cancer Res 20:4574-4583. Murine preB::300.19 CD38-expressing cells werecoated at 40,000 cells/well on 96-well High Bind plate (MSD L15XB-3) and100 μL/well of anti-CD38 antibodies were added for 45 min at 4° C. andwashed three times with PBS 1% BSA. 100 μL/well of goat anti-human IgGconjugated with Alexa488 (Jackson ImmunoResearch; #109-545-098) wasadded for 45 min at 4° C. and washed three times with PBS 1% BSA.Antibody binding was evaluated after centrifugation and resuspension ofcells by adding 200 μl/well PBS 1% BSA and read using Guava® easyCyte™8HT Flow Cytometry System. Apparent KD and EC50 values were estimatedusing BIOST@T-BINDING and BIOST@T-SPEED software, respectively.

Results

Binding of newly isolated mAb1 to human SU-DHL-8 or MOLP-8 cells wascompared to that of isatuximab using flow cytometry (FIG. 1A). Bothantibodies exhibited high-affinity binding to both cell lines. However,only mAb1 was able to bind to cells expressing cynomolgus monkey CD38 ontheir surface (FIG. 1B).

Binding to soluble extracellular domains of human and cynomolgus monkeyCD38 polypeptides was examined for newly isolated antibodies mAb1 andmAb6 using surface plasmon resonance (SPR). The SPR results aresummarized in Table A.

TABLE A Binding affinity of antibodies to the soluble extracellulardomain of hCD38 and cCD38 as determined by SPR assay. hCD38-hiscCD38-his (SEQ ID NO: 2) (SEQ ID NO: 4) Kd (s−1) KD (M) Kd (s−1) KD (M)mAb1 2.66E−04 3.36E−10 9.85E−05 3.90E−10 mAb6 2.03E−04 1.44E−09 1.90E−041.38E−09

Flow cytometry was also used to examine binding of mAb1 and mAb6anti-CD38 antibodies to murine pre-B cells expressing human orcynomolgus monkey CD38 polypeptide on their cell surface. The resultsare shown in Table B. Both antibodies tested exhibited high affinitybinding to preB::300.19 huCD38- or cCD38-expressing cells.

TABLE B Binding affinity of antibodies to hCD38 or cCD38 expressed bymurine preB::300.10 cells as determined by flow cytometry. Apparent KDFACS (M) hCD38-expressing cells cCD38-expressing cells mAb1 2.80E−102.20E−10 mAb6 2.07E−09 1.14E−09

These results demonstrate the generation of antibodies that bind withhigh affinity to both human and cynomolgus CD38 polypeptides. Theseantibodies, unlike other anti-CD38 antibodies, cross-react with humanand cynomologus CD38 polypeptides in soluble extracellular form orexpressed on the surface of mammalian cells.

Example 2: In Silico Design of Humanized Anti-CD38 Variants

This Example describes the humanization of antibodies generated inExample 1.

Sequences of mAb1 variable domains were analysed in-silico. TheInternational ImMunoGeneTics information system for immunoglobulins(IMGT) definition was used to identify Complementarity DeterminingRegions (CDRs).

First, in parallel, searches were undertaken to identify the closestMouse germline and Human germline protein sequence combination for eachvariable chain. This was performed using a Basic Local Alignment SearchTool (BLAST) (Nucleic Acids Res. 25:3389-3402) search against Mouse andHuman germline protein sequence databases. For each antibody chain,closest V and J mouse and human protein sequences were found. Thequalification of this high protein sequence identity was measured by theV region identity percentage. Results of the searches for mAb1 light andheavy chain are presented in Tables C and D, respectively.

TABLE C mAb1 variable light chain closest human and mouse (V&J) germlineprotein sequences. V Region Species V Allele Identity (%) J Allele [Homosapiens] IGKV4-1*01 (imgt) 66.34% IGKJ1*01 (imgt) Best FunctionalFunctional Functional Germlines [Mus musculus] IGKV3-10*01 (imgt) 96.97%IGKJ1*01 (imgt) Best Functional Functional Functional Germlines

TABLE D mAb1 variable heavy chain closest human and mouse (V&J) germlineprotein sequences. V Region Species V Allele Identity (%) J Allele [Homosapiens] IGHV1-3*01 (imgt) 69.39% IGHJ4*01 (imgt) Best FunctionalFunctional Functional Germlines [Mus musculus] IGHV1-12*01 (imgt) 87.76% IGHJ3*01 (imgt) Best Functional Functional Functional Germlines

Next, mAb1 variable domain sequences were screened for sequenceliabilities, such as deamidation, acidic cleavage, oxidation, oriso-aspartate formation sites. mAb1 structure has been analysed by 3Dmodelling by homology in order to define how amino acid residuesinteract in intra or intermolecular ways. This step led to theidentification of a group of amino acid residues that are structurallyimportant for mAb1 functionalities as CDRs conformation and antigenbinding. These amino acid residues were selected to be present in thehumanized mAb1 variable sequences.

CDRs from the parental murine mAb1 were grafted onto the relevantframeworks. Based on the above analyses, human IGKV3-20*02 coupled withIGKJ1*01 and human IGHV1-3*01 coupled with IGHJ4*01 were selected to befoundation for mAb1 variable light chain and variable heavy chainhumanization, respectively. The mAb1 light chain displayed 64.52%identity over the V region with the selected human IGKV3-20*02 germline.The mAb1 heavy chain displayed 69.39% identity over the V region withthe selected human IGHV1-3*01 germline. During humanization process, theparental murine mAb1 CDRs were transplanted between selected germlineframeworks in order to recompose standard antibody variable sequences.Attention was provided to the previously identified group of mAb1 aminoacid residues that are structurally important for its functionalities,as noted above. If needed those amino acid residues were replaced in thenewly created sequences by their exact corresponding mAb1 residue. Thiscorresponds to a back-mutation step to incorporate adequate parentalsequence amino acid residues. Some CDR mutations were incorporated forboth humanizing and avoiding sequence liabilities in parental CDRs. Thenewly created light and heavy variable sequences were used to generate3D homology models of the humanized mAb1 variable region. The 3D modelswere built using Model Antibody Framework from BIOVIA Discovery Studiosuite.

Based on CDR grafting approach, two variants for the variable lightchain (VL1 and VL3) and four variants for the variable heavy chain (VH1,VH3, VH5 and VH6) were generated. The particular combination of aminoacid residues which vary between mAb1 variable light and heavy sequencesand their humanized versions are set forth in Tables E and Table F,respectively.

TABLE E Sequence differences between mAb1 variable light chain andhumanized variants. Ig Regions parental humanized humanized (IMGT) mAb1VL VL1 VL3 FR1 S10 T T A12 S S V13 L L L15 P P Q17 E E CDR1 E27 Q E D30S D N34 Q N FR2 K49 R R CDR2 L54 G L FR3 N57 S S L58 R R E59 A A S60 T TV62 I I R72 G G D80 S S V82 L L A84 P P D85 E E A87 F F T89 V V

TABLE F Sequence differences between mAb1 variable heavy chain andhumanized variants. Ig Regions parental mAb1 humanized humanizedhumanized humanized (IMGT) VH VH1 VH3 VH5 VH6 FR1 Q5 V V V V L11 V V V VR13 K K K K S14 P P S P M20 V V V M CDR1 F32 Y F F F N33 A N N N FR2 T40A A A A G44 R R G R CDR2 N55 Q N N N FR3 K65 Q Q Q Q K67 R R R R S76 A AA A Q82 E E E E I83 L L I 1 T87 R R R R S91 T T T T

The humanized VL1 variant with SEQ ID NO: 14 displays a total of 22mutations (18 in FRs and 4 in CDRs) compared to the parental VL ofmAb1sequence with SEQ ID NO: 6. This variant derived from frameworks ofhuman germlines IGKV3-20*02 coupled with IGKJ1*01 with 8 back-mutationsdone due to the risk of negative impact on mAb structure, CDRsconformation and therefore, on binding to its target. Four positions ofthe parental CDRs were mutated in order to increase humanization rate oravoid sequence liabilities.

The humanized VL3 variant with SEQ ID NO: 18 displays a total of 18mutations (18 in FRs) compared to the parental VL of mAb1sequence withSEQ ID NO: 6. This variant derived from frameworks of human germlinesIGKV3-20*02 coupled with IGKJ1*01 with 8 back-mutations done due to therisk of negative impact on mAb structure, CDRs conformation andtherefore, on binding to its target.

The humanized VH1 variant with SEQ ID NO: 13 displays a total of 17mutations (14 in FRs and 3 in CDRs) compared to the parental VH ofmAb1sequence with SEQ ID NO: 5. This variant derived from frameworks ofhuman germlines IGHV1-3*01 coupled with IGHJ4*01 with 11 back-mutationsdone due to the risk of negative impact on mAb structure, CDRsconformation and therefore, on binding to its target. Three positions ofthe parental CDRs were mutated in order to increase humanization rate oravoid sequence liabilities.

The humanized VH3 variant with SEQ ID NO: 17 displays a total of 14mutations (14 in FRs) compared to the parental VH mAb1sequence with SEQID NO: 5. This variant derived from frameworks of human germlinesIGHV1-3*01 coupled with IGHJ4*01 with 11 back-mutations done due to therisk of negative impact on mAb structure, CDRs conformation andtherefore, on binding to its target.

The humanized VH5 variant with SEQ ID NO: 21 displays a total of 11mutations (11 in FRs) compared to the parental VH of mAb1sequence withSEQ ID NO: 5. This variant derived from frameworks of human germlinesIGHV1-3*01 coupled with IGHJ4*01 with 14 back-mutations done due to therisk of negative impact on mAb structure, CDRs conformation andtherefore, on binding to its target.

The humanized VH6 variant with SEQ ID NO: 23 displays a total of 12mutations (12 in FRs) compared to the parental VH of mAb1sequence withSEQ ID NO: 5. This variant derived from frameworks of human germlinesIGHV1-3*01 coupled with IGHJ4*01 with 13 back-mutations done due to therisk of negative impact on mAb structure, CDRs conformation andtherefore, on binding to its target.

The resulting light and heavy humanized variable sequences were blastedfor sequence similarity against the Immune Epitope Data Base (IEDB)database ((PLos Biol (2005) 3(3)e91) www.iedb.org) to ensure that noneof the sequences contained any known B- or T-cell epitope listedtherein.

The complete amino acid variable sequences of mAb1and humanized lightand heavy variable domains are set forth in Table G. These humanizedlight and heavy variable domains have been combined to generate severalhumanized versions of parental variable domains of mAb1. mAb2 variabledomains correspond to the association of humanized VH1 combined withhumanized VL1. mAb3 variable domains correspond to the association ofhumanized VH3 combined with humanized VL3. mAb4 variable domainscorrespond to the association of humanized VH5 combined with humanizedVL3. mAb5 variable domains correspond to the association of humanizedVH6 combined with humanized VL3. The trispecific binding proteins shownin Table G are described in greater detail in Example 4.

The corresponding coding DNA sequences of the humanized VH and VLvariants described above were cloned into a mammalian expression vectorencoding respectively the human IgG1 or human Ck domains for transientexpression and purification as described in Example 1. The aminosequences of the full length humanized anti-CD38 variants derived frommAb1 are listed as mAb2 (heavy chain: HC1, SEQ ID NO:15; light chain:LC1, SEQ ID NO:16), mAb3 (heavy chain: HC3, SEQ ID NO:19; light chain:LC3, SEQ ID NO:20), mAb4 (heavy chain: HC5, SEQ ID NO:22; light chain:LC3, SEQ ID NO:20), and mAb5 (heavy chain: HC6, SEQ ID NO:24; lightchain: LC3, SEQ ID NO:20).

Example 3: Cross-Reactivity and Apoptosis Induction of Anti-CD38Antibodies

The humanized anti-CD38 variants generated in Example 2 were nextcharacterized for binding to human and cynomolgus CD38 polypeptides andinduction of apoptosis.

Materials and Methods

Apoptosis Induction Assay

Cells were incubated at 2×10⁵ cells/mL in complete medium (RPMI-1640,10% FBS, 2 mM L-glutamine) with 1.5 μg/mL (10 nM) of indicatedantibodies for 20 hours at 37° C. with 5% CO2. Cells were stained withAnnexinV-FITC in accordance with the manufacturer's instructions (LifeTechnologies). Samples were analyzed by flow cytometry on a BD FACSAria™flow cytometer with BD FACSDiva software for acquisition control anddata analysis (both BD Biosciences).

Results

Binding properties of selected anti-human CD38 antibodies produced asdescribed above were examined (FIGS. 2A-2H). Binding of antibodies tosoluble human CD38 and cynomolgus monkey CD38 was examined using ELISAand SPR. ELISA data were used to determine the EC50 of antibody bindingto human and cynomolgus monkey CD38 for humanized anti-CD38 antibodiesmAb2 (FIG. 2A), mAb3 (FIG. 2C), mAb4, mAb5 (FIG. 2E), and humananti-CD38 antibody mAb6 (FIG. 2I).

The binding of the humanized anti-CD38 variants or human anti-CD38 mAbto CD38 was also evaluated using the SPR assay described above. SPR datawere used to determine the K_(D) and k_(off) of antibody binding tohuman and cynomolgus monkey CD38 for humanized anti-CD38 antibodies mAb2(FIG. 2B), mAb3 (FIG. 2D), mAb4, mAb5 (FIG. 2F), and human anti-CD38antibody mAb6 (FIG. 2H). The binding data are summarized in Table K showthat all the anti-CD38 mAbs bind to CD38 with similar bindingcharacteristics.

TABLE K Binding affinity of anti-CD38 mAbs to the soluble extracellulardomain of humanCD38 and cynomolgusCD38 as determined by surface plasmonresonance assay. hCD38-his (SEQ ID NO: 2) cCD38-his (SEQ ID NO: 4) Kd(s-1) KD (M) Kd (s-1) KD (M) mAb1 2.66E−04 3.36E−10 9.85E−05 3.90E−10mAb2 3.90E−04 3.32E−10 7.84E−04 3.44E−09 mAb3 2.83E−04 4.83E−10 1.29E−047.10E−10 mAb4 5.29E−04 8.22E−10 2.01E−04 1.14E−09 mAb5 3.33E−04 3.12E−101.25E−04 5.63E−10 mAb6 2.03E−04 1.44E−09 1.90E−04 1.38E−09

The ability of the humanized anti-CD38 variants to bind toCD38-expressing cells was assessed using the FACS-based binding assaydescribed above. FACS data were used to determine the EC50 of antibodybinding to human and cynomolgus monkey CD38 for humanized anti-CD38antibodies mAb2 (FIG. 2A), mAb3 (FIG. 2C), mAb4, mAb5 (FIG. 2E), andhuman anti-CD38 antibody mAb6 (FIG. 2G). The binding data, set forth inTable L, shows that all humanized anti-CD38 variants exhibited similarbinding affinities for cell surface CD38.

TABLE L Binding affinity of anti-CD38 mAbs to CD38 expressing murinepreB::300.19 cells. Apparent KD FACS (M) hCD38-expressing cellscCD38-expressing cells mAb1 2.80E−10 2.20E−10 mAb2 3.30E−10 7.50E−10mAb3 7.80E−10 1.31E−09 mAb4 5.50E−10 1.15E−09 tnAb5 6.80E−10 1.07E−09mAb6 2.07E−09 1.14E−09

Binding data from the three assays are summarized in FIG. 2I, along withsequence identity of the VH and VL domains to human V regions.

The abilities of parental mAb1 antibody and mAb7 to induce apoptosis wasnext examined. Both antibodies increased Annexin V staining andpropidium iodide (PI) uptake. 40% of cells became double-positive forAnnexin V and PI after treatment with mAb7, whereas 60% of cells treatedwith mAb1 were double positive. Both antibodies exhibited a similarconcentration-dependent apoptotic effect in SU-DHL-8 cells (FIG. 2J).Similarly, both antibodies promoted ADCC activity against SU-DHL-8 cellsin the presence of NK92 cells (FIG. 2K), leading to up to 60%cytotoxicity and an IC50 of 4-6 pM after 4 hours at 37° C. (FIG. 2L).

A CD38 isoform E was identified in silico and validated at thetranscript level from NK cells, PBMCs, and BMMCs from multiple myelomapatients and cancer cell lines (MOLP-8, CU1702, and CU2332). CD38isoform E was evidenced by a nucleic sequence database screening usingBLASTN 2.2.26 (Altschul, Stephen F., Thomas L. Madden, Alejandro A.Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman(1997), “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Res. 25:3389-3402.) program onHuman RefSeq transcript database release 20131216. BLASTN program wasapplied without masking sequence low complexity regions and consideringat least: 98.5% identity with human CD38 isoform A nucleic sequence on astretch of 100 nucleic acid residues long minimum. Sequences highlightedfrom this screening were realigned with CD38 gene locus in order to bevalidated as being transcriptional forms of Human CD38 gene (sameIntron-Exon genomic structure). CD38 isoform E nucleic sequence was oneof sequences validated as being transcriptional forms of the human CD38gene.

The ability of anti-CD38 antibodies to bind to both human CD38 isoformsA and E was also examined. For evaluating binding to CD38 isoform A andisoform E, an Enzyme-linked immunosorbent assay (ELISA) was performed byusing isoform A and isoform E proteins (prepared as described inExample 1) as capturing antigen. 96-well plates were coated with eitherisoform at 0.5 μg/well in PBS and 100 μL/well of antibodies were addedto the plate. The plate was incubated at 37° C. for 1 h and washed fivetimes with PBS containing 0.05% Tween-20 (PBS-T). Then, 100 μL of a1:25,000 dilution of Anti-human IgG, conjugated with horseradishperoxidase. (Jackson Ref: 109-035-098) was added to each well. Followingincubation at 37° C. for 1 h in darkness, plates were washed with PBS-Tfive times. Antibody binding was visualized by adding TMB-H2O2 bufferand read at a wavelength of 450 nm. EC50 values were estimated usingBIOST@T-SPEED software.

The binding affinity of various antibodies to CD38 isoform A (SEQ IDNO:1) and isoform E (SEQ ID NO:105) was determined, as shown in TableL2. Table M provides a comparison of binding properties for variousanti-CD38 antibodies.

TABLE L2 Binding affinity of anti-CD38 antibodies for CD38 isoforms Aand E, based on EC50 as determined by ELISA. Antibody CD38 isoform AEC50 (nM) CD38 isoform E EC50 (nM) mAb1 0.11 (CV 9%) 0.08 (CV 7%) mAb20.14 (CV 13%) 0.10 (CV 12%) mAb6 0.47 (CV 3.7%) 0.32 (CV 5%) mAb7 0.10(CV 7.1%) No binding

TABLE M Binding characteristics of various anti-CD38 antibodies. H11Anti- (Santa CD38 Cruz) Daratumumab mAb7 mAb1 mAb6 Binding to + + + + +huCD38 isoform A Binding to + − − + + huCD38 isoform E Binding to + −− + + cyno CD38

In conclusion, both mAb7 and mAb1 induce similar apoptosis in MOLP-8human multiple myeloma cells, similar concentration-dependent apoptoticeffect against SU-DHL-8 cells, and similar concentration-dependent ADCCactivity against SU-DHL-8 cells. However, only mAb1 bound to both humanand cynomolgus monkey CD38 with sub-nanomolar affinity and bound to CD38isoforms A and E.

The ability of the humanized anti-CD38 variants to induce apoptosis wasalso assessed by fluorescence activated cell sorter (FACS) as describedabove. Several anti-CD38 antibodies with various functional propertieshave been identified previously, while some of these can mediate invitro killing of CD38+ cell lines via direct impact on cellproliferation or apoptosis. The results of apoptosis induction assays asshown in FIG. 2M. All antibodies generated in Examples 1 and 2 were ableto induce apoptosis except mAb6. Anti-CD38 antibodies mAb2, mAb3, mAb4,and mAb5 led to a dose-dependent induction of apoptosis in SU-DHL-8lymphoma cells; IC50 for each antibody is provided in FIGS. 2N-2Q.

Example 4: Generation of Trispecific Anti-CD38 Binding Proteins

Next, binding properties of the antigen binding domains of selectedanti-CD38 antibodies described in Examples 1-3 were analyzed in thetrispecific format shown in FIG. 3A.

Anti-CD38 antigen binding domains were tested in trispecific format(anti-CD38xanti-CD28xanti-CD3) for ability to bind CD38 when otherantigen binding domains are bound to their cognate ligands using SPR.The sequential ligand binding assay is shown in FIG. 3B. For sequentialbinding of the three antigens to each trispecific Ab, saturatingconcentration (>10 KD) of each antigen was injected for 8 min followedby 5 min dissociation. Surface regenerate was conducted by injecting 10mM Glycine-HCl pH 2.5 for 60 sec at 30 μl/min. Data were fitted with 1:1kinetic binding model and analyzed using Biacore S200 EvaluationSoftware v 1.0. Equilibrium dissociation constant (K_(D)) was calculatedusing association rate constant (k_(on)) and dissociation rate constant(k_(off)).

As shown in FIG. 3C, this SPR-based assay showed that trispecificbinding proteins were able to bind CD38 regardless of whether the CD3and/or CD28 antigen binding domains were also bound to their cognateantigen. Results of an exemplary sequential binding assay are shown inFIG. 4. Kinetic parameters as measured by SPR are provided in Table M2.

TABLE M2 Binding of trispecific anti-CD38xanti- CD28xanti-CD3 bindingproteins to 1, 2, or 3 cognate antigens. Binding protein state prior toCD38 binding k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) No prebound 9.02E+051.42E−03 1.57E−09 Prebound CD3 8.35E+05 1.24E−03 1.48E−09 Prebound CD287.39E+05 1.32E−03 1.79E−09 Prebound CD3 then CD28 8.18E+05 1.23E−031.50E−09 Prebound CD28 then CD3 8.37E+05 1.23E−03 1.47E−09

These results demonstrate that all three targets can bind to thetrispecific binding proteins simultaneously. Pre-binding the trispecificbinding proteins with CD28, CD3, or both (in either order) did not alterbinding kinetics or binding affinity to CD38.

Next, each antigen binding domain of theCD38_(SB19)xCD28_(sup)xCD3_(mid) trispecific binding protein wasevaluated by SPR for the ability to bind cognate antigen with andwithout the other two antigen binding domains in saturation. Tables M3and M4 show the results of these assays.

TABLE M3 Target binding without other targets present Target k_(a)(M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) CD38 8.04E+05 1.41E−03 1,75E−09 CD281.16E+05 3.14E−04 2.71E−09 CD3 2.90E+04 6.73E−04 2.32E−08

TABLE M4 Target binding with other targets in saturation Target k_(a)(M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) CD38 5.93E+05 1.44E−03 2.42E−09 CD281.05E+05 3.96E−04 3.77E−09 CD3 1.27E+05 2.36E−03 1.86E−08

As demonstrated in Tables M3 and M4, having two targets saturated bypre-binding with antigen did not impact the kinetics or binding affinityof the third target for CD38 or CD28. In the case of CD3 binding,prebound CD38 and/or CD28 resulted in faster kinetics (approximately4-fold impact on k_(on) and k_(off) values).

Anti-CD38 antigen binding domains were tested in trispecific format withtwo anti-CD28 antigen binding domains (super agonist, “sup,” andconventional agonist, “cvn”) and two anti-CD3 antigen binding domains(“mid” and “low”). Variable domain sequences for these antigen bindingdomains are provided as follows: anti-CD28_(sup): SEQ ID NO:49 (VH) andSEQ ID NO:50 (VL); anti-CD28_(cvn): SEQ ID NO:51 (VH) and SEQ ID NO:52(VL); anti-CD3_(mid): SEQ ID NO:53 (VH) and SEQ ID NO:54 (VL);anti-CD3_(low): SEQ ID NO:84 (VH) and SEQ ID NO:85 (VL). The results ofSPR assays examining binding of trispecific binding proteins are shownin FIG. 5. Three anti-CD38 binding domains had roughly the same bindingaffinity in the trispecific binding protein format as in a monospecificformat. Both CD3 binding domains had approximately the same bindingaffinity in mono-, bi-, and trispecific formats. CD28 binding domainsshould slightly lower (but still nanomolar) binding affinity in bi- ortrispecific format as compared with monospecific. When the other twoantigen binding domains were saturated, anti-CD38_(SB19) andanti-CD28_(sup) binding domains had similar binding affinities, comparedwith when the other two antigen binding domains are not bound toantigen. However, the anti-CD3_(mid) binding domain showed fasterkinetics when the other two antigen binding domains were saturated.These results demonstrate that anti-CD38, anti-CD28, and anti-CD3binding domains are compatible for use with the trispecific bindingprotein format.

The anti-CD38 antigen binding domains generated herein were alsocompared against the existing anti-CD38 antigen binding domain of mAb7(see SEQ ID NO:47 for VH and SEQ ID NO:48 for VL sequences,respectively). The binding of trispecific molecules to CD38 expressed onthe surface of recombinant murine preB::300.19 cells was determined byflow cytometry and the corresponding anti-CD38 monovalent antibodieswere assayed in parallel. The recombinant cell line was described by J.Deckket et al. 2014 Clin. Cancer Res 20:4574-4583. Murine preB::300.19CD38-expressing cells were coated at 40,000 cells/well on 96-well HighBind plate (MSD L15XB-3) and 100 μL/well of trispecific molecules wereadded for 45 min at 4° C. and washed three times with PBS 1% BSA. 100μL/well of goat anti-human IgG conjugated with Alexa488 (JacksonImmunoResearch; #109-545-098) was added for 45 min at 4° C. and washedthree times with PBS 1% BSA. Antibody binding was evaluated aftercentrifugation and resuspension of cells by adding 200 μl/well PBS 1%BSA and read using Guava® easyCyte™ 8HT Flow Cytometry System. ApparentKD and EC50 values were estimated using BIOST@T-BINDING andBIOST@T-SPEED software, respectively.

Flow cytometry was used as described above to examine binding of mAb7 orthe trispecific binding protein with the mAb7 anti-CD38 antigen bindingdomain to murine pre-B cells expressing human or cynomolgus monkey CD38polypeptide on their cell surface. As shown in FIG. 6A, theCD38xCD28_(sup)xCD3_(mid) trispecific binding protein with the mAb7anti-CD38 antigen binding domain bound to cells expressing human CD38(upper left) with 8-fold lower apparent affinity than mAb7 monospecificantibody (upper right). Neither mAb7 monospecific antibody (lower right)or the trispecific binding protein with the mAb7 anti-CD38 antigenbinding domain (lower left) bound to cells expressing cynomolgus CD38.

The binding domain of humanized anti-CD38 antibody mAb2 was also testedin trispecific formats for binding to cells expressing human orcynomologus CD38 polypeptides. As shown in FIGS. 6B-6D, and unlike mAb7,CD38xCD28_(sup)xCD3_(mid) and CD38xCD28_(cvn)xCD3_(mid) trispecificbinding proteins with mAb2 anti-CD38 antigen binding domain, as well asthe mAb2 monospecific antibody, were able to bind both human andcynomolgus monkey CD38 polypeptides. The CD38xCD28_(cvn)xCD3_(mid)trispecific binding protein with the mAb2 anti-CD38 antigen bindingdomain bound to cells expressing human CD38 with 9-fold lower apparentaffinity than the parental mAb2 antibody (compare FIG. 6C, top to FIG.6D, top). The CD38xCD28_(cvn)xCD3_(mid) trispecific binding protein withthe mAb2 anti-CD38 antigen binding domain bound to cells expressingcynomolgus CD38 with 7.5-fold lower apparent affinity than the parentalmAb2 antibody (compare FIG. 6C, bottom to FIG. 6D, bottom). TheCD38xCD28_(sup)xCD3_(mid) trispecific binding protein with the mAb2anti-CD38 antigen binding domain bound to cells expressing human CD38with a 2.5-fold lower apparent affinity than theCD38xCD28_(cvn)xCD3_(mid) trispecific binding protein with the mAb2anti-CD38 antigen binding domain (compare FIG. 6C, top to FIG. 6B, top).

The binding domain of humanized anti-CD38 antibody mAb6 was alsocompared against the mAb6 monospecific antibody for binding to cellsexpressing human or cynomologus CD38 polypeptides. While the mAb6monospecific antibody bound to cells expressing human (upper right) orcynomolgus monkey (lower right) CD38 polypeptides in the nM range (FIG.6E), the CD38xCD28_(sup)xCD3_(mid) trispecific binding protein with themAb6 anti-CD38 antigen binding domain bound to cells expressing human(upper left) or cynomolgus monkey (lower left) CD38 polypeptides withoutsaturation.

In conclusion, the affinity for CD38_(SB19)xCD28_(sup)xCD3_(mid)trispecific binding protein binding to human CD38 was found to be in thesame range, whether examining binding to recombinant human CD38 by SPRor to human CD38 expressed on a cell surface by flow cytometry (FIG.6F). Similarly, the affinity of CD38_(VH1)xCD28_(sup)xCD3_(mid/low)(mAb2 anti-CD38 binding domain) and CD38_(VH1)CD28_(cvn)xCD3_(mid/low)trispecific binding proteins (mAb2 anti-CD38 binding domain) for bindingto human CD38 was also in the same range in both assays. ForCD38_(HHY1370)xCD28_(sup)xCD3_(mid) (mAb6 anti-CD38 binding domain), theK_(D)for binding human CD38 was determined by SPR to be 1 nM, whereas noaccurate EC50 value could be estimated by flow cytometry. A summary ofapparent KD values (obtained by FACS analyses) of trispecific bindingproteins with various anti-CD38 binding domains is provided in Table M5.

TABLE M5 Summary of apparent KD values obtained by flow cytometry assaysApparent KD FACS (M) hCD38- cCD38- expressing cells expressing cellsTrispecific with  4.4 nM 7.5 nM antiCD38 mAb2 Trispecific with Nosaturation No saturation antiCD38 mAb6 Trispecific with   4 nM Nobinding antiCD38 mAb7 mAb2  0.5 nM   1 nM mAb6 11.2 nM  66 nM mAb7  0.5nM No binding

As expected, ΔCD38xCD28_(sup)xCD3_(mid) trispecific binding proteinlacking the anti-CD38 binding domain did not bind to cells expressinghuman or cynomolgus monkey CD38 polypeptides (FIG. 6G). This indicatesthat the binding observed in this assay was specific for the CD38antigen binding domains.

Example 5: In Vitro and In Vivo Characterization of Anti-CD38 mAb2 andAnti-CD38 mAb6-Containing Trispecific Binding Proteins

The following Example describes experiments characterizing thestability, binding properties, and activities of novel T cell engagersthat contain variable domains derived from the mAb2 and mAb6 antibodies.Additional anti-CD38 x CD28 x CD3 antibodies were generated comprisingvariants of the anti-CD38, CD3 and CD28 arms of the trispecific bindingproteins. The new anti-CD38/CD3/CD28 antibodies differ in: 1) Anti-CD38binding domain (mAb2 or mAb6); 2) Anti-CD3 binding domain (CD3_(high) orCD3_(low); see SEQ ID NOs: 84 and 85 for anti-CD3_(low) VH and VLsequences, respectively); 3) Anti-CD28 binding domain (CD28_(sup) orCD28_(cvn)). Within possible combinations, a collection of the anti-CD38x CD28 x CD3 was designed, produced, and subsequently tested for variousfunctions.

Materials and Methods

Production and Purification of Trispecific Binding Proteins

Trispecific binding proteins were produced by transient transfection of4 expression plasmids into Expi293 cells using ExpiFectamine™ 293Transfection Kit (Thermo Fisher Scientific) according to manufacturer'sprotocol. Briefly, 25% (w/w) of each plasmid was diluted into Opti-MEM,mixed with pre-diluted ExpiFectamine reagent for 20-30 minutes at roomtemperature (RT), and added into Expi293 cells (2.5×10⁶ cells/ml). Anoptimization of transfection to determine the best ratio of plasmids wasoften used in order to produce the trispecific binding protein with goodyield and purity.

4-5 days post transfection, the supernatant from transfected cells wascollected and filtered through 0.45 μm filter unit (Nalgene). Thetrispecific binding protein in the supernatant was purified using a3-step procedure. First, protein A affinity purification was used, andthe bound Ab was eluted using “IgG Elution Buffer” (Thermo FisherScientific). Second, product was dialyzed against PBS (pH7.4) overnightwith 2 changes of PBS buffer. Any precipitate was cleared by filtrationthrough 0.45 μm filter unit (Nalgene) before next step. Third,size-exclusion chromatography (SEC) purification (Hiload 16/600 Superdex200 pg, or Hiload 26/600 Superdex 200 pg, GE Healthcare) was used toremove aggregates and different species in the prep. The fractions wereanalyzed on reduced and non-reduced SDS-PAGE to identify the fractionsthat contained the monomeric trispecific binding protein beforecombining them. The purified antibody can be aliquoted and stored at−80° C. long term.

ELISA Assays

The binding properties of the purified antibodies were analyzed eitherusing ELISA or SPR methods. For ELISA, corresponding antigens for eachbinding site in the trispecific binding protein were used to coat a96-well Immuno Plate (Nunc 439454, Thermo Fisher Scientific) overnightat 4° C. using 2 μg/ml each antigen in PBS (pH7.4). The coated plate wasblocked using 5% skim milk+2% BSA in PBS for one hour at RT, followed bywashing with PBS+0.25% Tween 20 three times (Aqua Max 400, MolecularDevices). Serial dilution of antibodies (trispecific and control Abs)were prepared and added onto the ELISA plates (100 μl/well induplicate), incubated at RT for one hour, followed by washing 5 timeswith PBS+0.25% Tween 20.

After washing, the HRP conjugated secondary anti-human Fab (1:5000, Cat.No. 109-035-097, Jackson ImmunoResearch Inc) was added to each well andincubated at RT for 30 minutes. After washing 5 times with PBS+0.25%Tween 20, 100 μl of TMB Microwell Peroxidase Substrate (KPL,Gaithersburg, Md., USA) was added to each well. The reaction wasterminated by adding 50 μl 1M H₂SO₄, and OD₄₅₀was measured usingSpectraMax M5 (Molecular Devices) and analyzed using SoftMax Pro6.3software (Molecular Devices). The final data was transferred to GraphPadPrism software (GraphPad Software, CA, USA), and plotted as shown. EC50was calculated using the same software.

ELISA assay was used to determine the binding of an anti-CD38xCD28xCD3trispecific antibodies or isotype control antibody (human IgG4) to humanCD3 (Cambridge Biologics LLC Cat #03-01-0051), CD28 (Cambridge BiologicsLLC Cat #03-01-0303), and CD38 (Cambridge Biologics LLC Cat#03-01-0369). The bound antibodies were detected using a horseradishperoxidase (HRP)-conjugated anti-Fab secondary antibody (JacksonImmunoResearch Inc #109-035-097).

In Vitro Cell Killing Assay

Purified human PBMCs were using for in vitro killing assays againstvarious cancer cells using different trispecific binding proteins.Briefly, the killing assay was set up in 96-well V-bottom plate. Foreach plate, 40 ml PBMCs from each donor were plated at 2×10{circumflexover ( )}6 cells/ml, and 30 ml of PKH26 (Sigma #MINI26) labeled targetcells at 2.5×10{circumflex over ( )}5 cells/ml (4 μL of dye to stain upto 1×10{circumflex over ( )}7 cells) were prepared. First 20 μL/welltest proteins at various concentrations or PMA were added into eachwell, followed by adding 80 μL/well labeled target cells into each well(2×10{circumflex over ( )}4 cells/well). 100 μL of PBMC were then addedto each well, reaching E:T=10:1 well (2×10{circumflex over ( )}5cells/well), and incubated for 24 hours at 37° C. 5% CO2 incubator. Thecells were spin down, and the supernatant was either collected formeasuring cytokine release, or discarded. The cells were stained withVivid LIVEDEAD™ Fixable Violet Dead Cell Staining buffer (LifeTechnology #L34955) (staining buffer was prepared by adding 60 μL Vividreagent into 60 ml PBS). Cells were resuspended into 100 μL stainingbuffer by incubation for 15 min at RT in the dark. After washing thecells with 1×PBS, the cells were resuspended in 200 μL PBS with 0.5%Paraformaldehyde, and PKH26+Vivid+ cancer cells were collected byFortessa flow cytometer (Beckton Dickinson, San Jose, Calif.), followedby analysis using the Flowjo software. The percentage of killing iscalculated as specific killing-spontaneous killing/total cells andplotted as shown.

Cytokine Release Assay

For measuring inflammatory cytokine concentrations in the in vitroactivation assays, in vitro killing assays, in vivo activation assays inCD34+ umbilical cord cell humanized NSG mice, and the toxicity study,cell culture supernatant was collected, and serum samples were dilutedaccording to manufacturer's protocol using Milliplex Human HighSensitivity T cell 13-plex Kit (EMD Millipore). These were subsequentlyanalyzed by EMD Millipore MAGPIX® System, and MILLIPLEX® Analyst 5.1software.

In Vivo Mouse Models and Efficacy Studies

Human CD34+ hematopoietic stem cell-engrafted NSG mice (hu-CD34) wereused as an in vivo mouse model. These mice develop multi-lineage humanimmune cells, and are a validated platform for immuno-oncology efficacystudies (see, e.g., Shultz, L. D. et al. (2014) Cold Spring Harb.Protoc. 2014:694-708). Hu-CD34⁺ NSG mice are produced by injecting CD34⁺hematopoietic stem cells, showing effective multi-lineage engraftment ofhuman immune cell populations including T cells, B cells and some otherpopulations (McDermott, S. P. et al. (2010) Blood 116:193-200).Multi-lineage hematopoiesis occurs within 12 weeks. Engraftment isstable for over one year without graft-versus-host disease.

For the efficacy study using hu-CD34 NSG mice, mice were purchased fromThe Jackson Laboratory (Maine, USA), and human cell populations werevalidated before use. In general, 5×10⁶ tumor cells mixed in Matrigel(BD Biosciences) (50% v/v) were used for inoculating tumor in eachmouse. Once tumor size reached the range of 100-150 mm³, mice wereselected and randomized into each group for study. Antibodies were givenintravenously at given doses 3 times weekly. Body weight was monitored1-3 times weekly. Tumor size was measured by caliper tumor measurements1-3 times/week. All mice were terminated when the tumor size reached1,500 mm³, or 24 hours after the last dose. Terminal blood samples (0.3mL) were collected into serum separator tubes, mixed by gently invertingfive times, and placed into a tube rack. Terminal tumors were alsocollected and weighed before being put into fixative forimmunohistochemistry analysis.

Human PBMC humanized (hu-PBMC) NSG mice were used as another in vivomouse model. These mice are produced by injecting purified human PBMCfrom health donors, which have the fastest engraftment rate using adultperipheral blood mononuclear cells and enable short-term studiesrequiring a strong effector and memory T cell and NK cell function, andare suitable for short term efficacy study (3-4 weeks) due tograft-versus-host disease.

For the efficacy study using hu-PBMC NSG mice, 8-10 week old NSG mice(Cat. No: 005557, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were purchased fromThe Jackson Laboratory (Maine, USA). Each mouse was innoculated with5×10⁶ tumor cells mixed in Matrigel (BD Biosciences) (50% v/v)subcutaneously. Once tumor size reached the range of 50-100 mm³, 10×10⁶human PBMCs from healthy donors were reconstituted to each mouseintraperitoneally (IP). Human cell reconstitution was validated the nextday. Once tumor size reached the range of 100-150 mm³, mice wereselected and randomized into each group for study. Antibodies were givenintravenously at given doses 3 times weekly. Body weight was monitored1-3 times weekly. Tumor size was measured by caliper tumor measurements1-3 times/week. All mice were terminated when the tumor size reached1,500 mm³ or 24 hours after the last dose. Terminal blood samples (0.3mL) were collected into serum separator tubes, mixed by gently invertingfive times, and placed into a tube rack. Terminal tumors were alsocollected and weighed before being put into fixative forimmunohistochemistry analysis.

For disseminated hu-PMBC NSG mouse model, 1-5×10⁶ cells were injectedinto each mouse intravenously at day 0. At day 3, base line luminanceimaging was taken for randomization. At day 4, 10×10⁶ human PBMCs fromhealthy donors were reconstituted to each mouse intraperitoneally (IP).Mice were treated weekly at day 5, 12, 19 using indicated doses. Weeklyluminance body imaging was taken on day 10, 17, 24 for monitoring tumorvolume in each animal. At termination of the study, blood, spleen, boneand bone marrow were collected for histopathology study.

Results

The ability of the antibodies to bind all three target antigens wastested by ELISA assay. CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4,CD38_(VH1)xCD28_(sup)xCD3_(low) IgG4, CD38_(VH1)xCD28_(cvn)xCD3_(mid)IgG4, CD38_(VH1)xCD28_(cvn)xCD3_(low) IgG4 showed similar bindingaffinity to human and monkey CD3 and CD38, but variable binding affinityto CD28 (human and monkey have identical extracellular domain) withCD28_(sup) showing better affinity (FIG. 7A).CD38_(VH1)xCD28_(sup)xCD3_(mid) and CD38_(HHY1370)xCD28_(sup)xCD3_(mid)IgG4 FALA variant and IgG1 LALA P329A and NNAS variants all showedsimilar binding to human CD3, CD28, and CD38 (FIG. 7B).

Next, the trispecific anti-CD38xCD3xCD28 binding protein variants weretested for their capability of inducing T cell activation andantibody-mediated tumor cell killing (FIGS. 8A-8D). FIG. 8A shows the invitro killing activities of CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4,CD38_(hhy1370)xCD28_(sup)xCD3_(mid) IgG4,CD38_(VH1)xCD28_(cvn)xCD3_(mid) IgG4,CD38_(hhy1370)xCD28_(cvn)xCD3_(mid) IgG4, against human multiple myelomacell line RPMI-8226 using PBMC from 3 different donors at E:T=10. TheEC50 of the killing activity was calculated and summarized in Table N.Both anti-CD38xCD3xCD28 trispecific binding proteins containingCD28_(sup) showed better killing activities. The in vitro killingactivities of these molecules was also examined using NCI-H929 (FIG.8B), KMS-26 (FIG. 8C), and KMS-11 cell lines. (FIG. 8D).

Anti-CD38xanti-CD3xanti-CD28 trispecific binding proteins with IgG1 LALAP329A and NNAS variants, or IgG4 FALA variant, also displayed in vitrokilling of multiple myeloma KMS-11 (FIG. 8E) and U266 (FIG. 8F) cells.The EC50 for each antibody against each cell line was calculated andsummarized in Table Q2 (KMS-11) and Q3 (U266).

FIGS. 9A, 91B, & 10 show the in vitro T cell activation profile of theCD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 and CD38_(VH1)xCD28_(sup)xCD3_(low)IgG4. Both antibodies showed similar activation activities for CD4 andCD8 T cells. The EC5 for each antibody against each cell line wascalculated and summarized in Table N (RPMI-8226), Table O (NCI-H929),Table P (KMS-26), and Table Q (KMS-11).

TABLE N Antibody-mediated specific killing of CD38+ RPMI8266 cells byPBMCs from different donors EC50 (ng/ml) Donor Donor Donor Antibody #65#67 #68 CD38_(VH1) × CD28_(sup) × CD3_(mid) IgG4 5.30 1.95 3.3CD38_(hhy1370) × CD28_(sup) × CD3_(mid) IgG4 2.24 3.42 3.4 CD38_(VH1) ×CD28_(cvn) × CD3_(mid) IgG4 8.31 29.52 46.8 CD38_(hhy1370) × CD28_(cvn)× CD3_(mid) IgG4 4.62 8.98 16.5

TABLE O Antibody-mediated specific killing of CD38+ NCI-H929 cells byPBMCs from different donors EC50 (ng/ml) Donor Donor Donor Antibody #65#67 #68 CD38_(VH1) × CD28_(sup) × CD3_(mid) IgG4 0.51 0.48 0.9CD38_(hhy1370) × CD28_(sup) × CD3_(mid) IgG4 0.94 1.84 2.5 CD38_(VH1) ×CD28_(cvn) × CD3_(mid) IgG4 3.70 9.47 10.4 CD38_(hhy1370) × CD28_(cvn) ×CD3_(mid) IgG4 2.31 6.39 7.2

TABLE P Antibody-mediated specific killing of CD38+ KMS-26 cells byPBMCs from different donors EC50 (ng/ml) Donor Donor Donor Antibody #65#67 #68 CD38_(VH1) × CD28_(sup) × CD3_(mid) IgG4 1.03 0.91 1.3CD38_(hhy1370) × CD28_(sup) × CD3_(mid) IgG4 3.95 5.06 5.3 CD38_(VH1) ×CD28_(cvn) × CD3_(mid) IgG4 10.73 28.17 22.4 CD38_(hhy1370) × CD28_(cvn)× CD3_(mid) IgG4 15.84 32.10 25.7

TABLE Q Antibody-mediated specific killing of CD38+ KMS-11 cells byPBMCs from different donors EC50 (ng/ml) Donor Donor Donor Antibody #65#67 #68 CD38_(VH1) × CD28_(sup) × CD3_(mid) IgG4 3.05 3.40 14.7CD38_(hhy1370) × CD28_(sup) × CD3_(mid) IgG4 4.17 7.74 25.1 CD38_(VH1) ×CD28_(cvn) × CD3_(mid) IgG4 18.14 98.83 566.8 CD38_(hhy1370) ×CD28_(cvn) × CD3_(mid) IgG4 16.30 27.63 139.2

TABLE Q2 Antibody-mediated specific killing of CD38 + KMS-11 cells byPBMCs from different donors (IgG1/4 variant Fcs) CD38_(VH1)- CD38_(VH1)-xCD28_(sup) ₋ CD38_(VH1)- CD38_(hhy1370)- xCD28_(sup)- xCD3_(mid)xCD28_(sup)- CD38_(hhy1370)- xCD28_(sup)- CD38_(hhy1370)- EC50xCD3_(mid) IgG1 xCD3_(mid) xCD28_(sup)- xCD3_(mi) xCD28_(sup)- (PM) IgG4LALA IgG1 xCD3_(mi) IgG1 LALA xCD3_(mi) (KMS11) FALA P329A NNAS IgG4FALA P329A IgG1 NNAS KP50901 3.879 4.375 4.411  7.731  9.311 17.71KP50904 4.379 6.739 8.644 19.01 18.88 24.39

TABLE Q3 Antibody-mediated specific killing of CD38 + U266 cells byPBMCs from different donors (IgG1/4 variant Fcs) CD38_(VH1)- CD38_(VH1)-xCD28_(sup) ₋ CD38_(VH1)- CD38_(hhy1370)- xCD28_(sup)- xCD3_(mid)xCD28_(sup)- CD38_(hhy1370)- xCD28_(sup)- CD38_(hhy1370)- EC50xCD3_(mid) IgG1 xCD3_(mid) xCD28_(sup)- xCD3_(mi) xCD28_(sup)- (PM) IgG4LALA IgG1 xCD3_(mi) IgG1 LALA xCD3_(mi) (U266) FALA P329A NNAS IgG4 FALAP329A IgG1 NNAS KP50901 1.879 1.031 1.150 2.691 1.597 2.816 KP509040.8657 3.570 2.018 2.112 0.8375 3.527

Cytokine production induced by the trispecific binding protein variantswas also examined (FIGS. 11A & 11B) using the method described inStebbings, R. et al. (2007) (J. Immunol. 179:3325-3331) by coating theplate with indicated antibodies, followed by incubation with human PBMCfor 24 hours using 2 concentrations of the testing antibodies (5 μg/mland 25 ng/ml). 24-hour culture supernatants were collected and used tomeasure the IL2, IL6, IL10, IL12, and TNF-α, IFN-γ concentration in thesupernatants as described above. CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4,CD38_(hhy1370)xCD28_(sup)xCD3_(mid) IgG4,CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4,CD38_(hhy1370)xCD28_(cvn)xCD3_(mid) IgG4 all stimulated production ofsignificant level of IL2, TNF-α, and IFN-γ at 5 μg/ml, but failed toinduce measurable level of any cytokine at 25 ng/ml, a dose showing invivo efficacy in 2 humanized NSG mouse models.

Anti-tumor activity was tested in vivo mouse models for the trispecificbinding protein variants (FIGS. 12A-13F) as described above. FIGS.12A-12E show the results of the in vivo efficacy study using the humanCD34+ hematopoietic stem cell-engrafted NSG mouse (hu-CD34) modelimplanted with human MM cell line RPMI-8226.CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 and bench marker control CD38xCD3bispecific antibodies were used to treat tumor bearing mice at indicateddoses 3QW (6 doses total). The body weight and tumor growth for eachmouse were measured and plotted (FIGS. 12A & 12E). Average tumor growthcurve (FIG. 12D), Day 18 tumor volume (FIG. 12B) and D19 terminal tumorweight (FIG. 12C) for each group were also plotted. All groups treatedwith CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 not only showed statisticalefficacy compared to PBS control, but also showed statistically betterefficacy at 1 μg/kg dose compared to control CD38xCD3 bispecificantibody.

FIGS. 13A-13F show the results of the in vivo efficacy study using thehuman PBMC humanized NSG mouse model implanted with human MM cell lineRPMI-8226. CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 and benchmark controlCD38xCD3 bispecific antibodies were used to treat tumor bearing mice atindicated doses 3QW (8 doses total). The tumor growth for each mouse wasmeasured and plotted (FIG. 13A). Average tumor growth curve (FIG. 13F),Day 4 tumor volume (day of starting the treatment; FIG. 13B), Day 21tumor volume (day of last treatment; FIG. 13C), Day 21 average tumorvolume (FIG. 13D) and Day 22 terminal tumor weight (FIG. 13E) for eachgroup were also plotted. All groups treated withCD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 not only showed statisticalefficacy compared to PBS control, but also showed statistically betterefficacy at multiple doses compared to control CD38xCD3 bispecificantibody, indicating superior in vivo anti-tumor activity by thetrispecific anti-CD38/CD3/CD28 antibody.

Benchmark control CD38xCD3 bispecific antibody comprised the followingsequences:

SEQ ID NO 108: benchmark heavy chain 1 (binds CD38)EVQLVESGGGLVQPGGSLRLSCAASGFTFSYSWMNWVRQAPGKGLEWVSEINPQSSTINYATSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGNWFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSDTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEEYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCDVSGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWEQGDVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO 109: benchmark heavy chain 2 (binds CD3)EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAYWGQGTLVTVSSGKPGSGKPGSGKPGSGKPGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIGGTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFGGGTKLTVLEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREQMTKNQVKLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO 110: benchmark light chain(binds CD38) DIVMTQSPSSLSASVGDRVTITCRASQNVDTWVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFATYFCQQYDSYPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Example 6: Dose Escalation Study with Anti-CD38 mAb2 and mAb6-ContainingTrispecific Binding Proteins

Materials and Methods

All NHP studies were carried out by Covance (Princeton, N.J., USA)according to Covance ICUCA protocol. Drug- and protein-naïve orprotein-naïve male Cynomolgus Monkeys were used in all studies. Based onstudy design, monkeys were selected and grouped for each trispecificbinding protein. Antibody was given by intravenous infusion for 1 hourvia saphenous vein. Increasing doses were given on consecutive days forlow doses (<10 μg/kg), but with a 1-2 day interval for higher doses (>10μg/kg) for observation purposes. Blood samples were collected at 0 hour(Day 1 only), 0.5 hour (mid-infusion), 1, and 6 hours from start ofinfusion for all animals after each dose, as specified. Additionalunscheduled blood samples were collected at the discretion of the studydirector, pathologist, and/or clinical veterinarian. All animals werereturned to colony on Day 60. PBMC and serum from the blood samples wereprepared using standard methods, and preserved for future analysis.

Blood from treated non-human primates was stained with fluorescentlyconjugated antibodies against T cell markers and human IgG, Fcγ fragmentand analyzed on a flow cytometer.

Results

A dose escalation toxicity study using mAb2-containingCD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4, mAb6-containingCD38_(hhy1370)xCD28_(sup)xCD3_(mid) IgG4, mAb2-containingCD38_(VH1)xCD28_(cvn)xCD3_(mid) IgG4, and mAb6-containingCD38_(hhy1370)xCD28_(cvn)xCD3_(mid) IgG4 trispecific binding proteinswas carried out in non-human primates. All three binding domains in 4binding proteins are cross-reactive with cynomolgus CD38/CD3/CD28polypeptides. The study was devised to assess the potential toxicityprofile of the molecular. Blood samples were collected for serum andPBMC isolations. Circulating T cell populations were investigated aftereach dosing (FIGS. 14E-14H), along with T cell subpopulation activation(CD69+) (FIGS. 14A-14D). Percentage of CD4 and CD8 T cells incirculation were decreased with dose escalation. Significant CD4 and CD8T cell activation were prominent starting at 2.5 μg/kg doses, suggestingpotent activation ability for mAb2- and mAb6-containing molecules.Significant circulating T cell deletion was seen with all proteins at12.5 μg/kg (FIGS. 14I-14L), again correlating with potency. Depletion ofcirculating T cells was rather transient, returning the pre-treatmentlevel after 24-48 hours (FIGS. 14M-14P). Serum level of severalcytokines was also measured. Significant IL-6 and IL-10 release wereobserved at 12.5 μg/kg with all molecules, which was rather transient,returning to baseline at 24 hours (FIG. 14U).

CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 (FIG. 14V) andCD38_(VH1)xCD28_(cvn)xCD3_(mid) IgG4 (FIG. 14W) both induced depletionof T cells in blood at higher doses. Similarly,CD38_(hhy1370)xCD28_(sup)xCD3_(mid) IgG4 (FIG. 14X) andCD38_(hhy1370)xCD28_(cvn)xCD3_(mid) IgG4 (FIG. 14Y) also induceddepletion of T cells in blood at higher doses. However, T cells startedto reappear in blood 24 hours after treatment with any of the fourtrispecific binding proteins (FIGS. 14Z-14AC). FIGS. 14AD & 14AE showthe amount of CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 bound to CD4+ T cellsafter administration of a 100 μg/kg dose. FIGS. 14AF & 14AG show theamount of CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 bound to CD8+ T cellsafter administration of a 100 μg/kg dose. These data clearlydemonstrated that trispecific Ab CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4can bind to T cells in vivo with prolonged time (48-72 hours).

Example 7: Optimization of Pharmacokinetics/Pharmacodynamics by FcVariants

Materials and Methods

C-terminal 6-His tagged recombinant human Fcγ RI (R&D Systems#1257-FC-050), C-terminal 10-His tagged recombinant human Fcγ RIIA (R&DSystems #1330-CD-050/CF) and C-terminal HPC4 tagged recombinant humanFcγRIII (V158 or F158) were captured to a Biacore chip. Antibodies at200, 100 & 50 nM were injected for 2 min, followed by 2 min dissociationin HBS-P+, 2 mM CaCl2) pH 7.4 buffer at 30 μL/min flow-rate usingBiacore 200. Binding curve at 200 nM was shown.

ELISA assay using C-terminal HPC4 tagged recombinant human Neonatal FcReceptor (FcRn) was used to measure the binding properties of thetrispecific binding proteins with different Fe modifications. Briefly,recombinant human Neonatal Fc Receptor (FcRn) was used to coat the ELISAplate (Nunc 80040LE)(2 ug/ml in PBS) overnight. Serial dilutedtrispecific binding proteins with different Fc modifications were addedinto each well and incubated at room temperature for one hour, followedby washing and incubating with HRP labeled anti-human IgG secondaryantibody.

Results

Variants of trispecific binding proteins described above were nextassayed for binding to various Fc receptors in order to optimizepharmacokinetics (PK)/pharmacodynamics (PD).

Human Fc variants were characterized for binding to FcγR I (FIG. 15A),FcγR IIa (FIG. 15B), and FcγR IIIb/c (FIG. 15C) as described above.Variants tested were human IgG1, human IgG4, and human IgG4 with FALAmutations (F234A and L235A according to EU index) with a controltrispecific binding protein.

As shown in FIGS. 15A-15C, wild-type IgG1 and IgG4 were able to bindFcγR I and FcγR IIa, but not FcγR IIIb/c, as previously reported. IgG4FALA mutations eliminated FcγR I and FcγR IIa binding. Without wishingto be bound to theory, it is thought that eliminating FcγR I and FcγRIIa binding can improve PK/PD by removing unintended clustering throughFc/FcγR interactions.

Next, the IgG4 FALA variant was examined for binding FcRn. The variant,as well as wild-type IgG4, bound to FcRn (FIG. 16). These resultsdemonstrate that the IgG4 FALA mutations do not affect the interactionsbetween IgG4 Fc and FcRn, implying a minimal impact on binding proteinhalf-life.

The PK parameters of CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4,CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 FALA,CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG1 LALA P329A, andCD38_(HHY1370)xCD28_(sup)xCD3_(mid) IgG4 FALA, as determined in NSGmice, are summarized in FIG. 17. IgG4 Fc modifications showedsignificantly improved half-life and AUC in NSG mice by eliminatingbinding to NSG-specific FcγRI, which is abundant on macrophages.

Example 8: In Vitro Activation and In Vivo Anti-Tumor Efficacy ofAnti-CD38 Trispecific Binding Proteins

Materials and Methods

Cytokine Release from Human PBMCs

Human PMBCs were incubated with 100 pM of binding proteins for 24 hrs.Supernatants were collected and cytokines measured by Luminex using DropArray plates (in triplicate). For experiments using tumor target cells,Human PMBC were incubated with 100 pM of antibodies and with, orwithout, RPMI-8226 target cells at 10:1 E:T ratio for 24 hrs.Supernatants were collected after PMBCs incubated with trispecificproteins and assayed for cytokines with MILLIPLEX® MAP kit and analyzedon a MAGPIX® System.

Bcl-xL Assay

Negatively sorted T cells from PBMC (magnetic) were incubated with 100nM plate-bound Abs for 1 day. T cells were then washed and stainedstained with fluorescently conjugated antibodies specific for T cellmarkers and Bcl-xL and analyzed on a flow cytometer.

T Cell Proliferation

Negatively sorted T cells from PBMC (magnetic bead cell separation) wereincubated with plate-bound Abs (100 nM) for 1 to 6 days. Total cellswere counted by flow cytometry using counting beads on specific daysafter start of incubation. Fold change was calculated from Day 0 cellcounts (500 cells/uL). Results from 3 PBMC donors.

IL-2 Expression

GloResponse™ IL2-luc2P Jurkat Cells were incubated with trispecificproteins for 6 hours, then Bio-Glo™ luciferase assay system was used todetect luciferase reporter gene expression.

PBMC In Vitro Activation

The activation (CD69⁺) of human PBMC cells treated withanti-CD38xCD28xCD3 trispecific antibodies or control IgG4 bindingprotein was determined.

In Vivo Efficacy in Disseminated Tumor Model

For disseminated hu-PMBC NSG mouse model, 1-5×106 cells were injectedinto each mouse intravenously at day 0. At day 3, base line luminanceimaging was taken for randomization. At day 4, 10×106 human PBMCs fromhealthy donors were reconstituted to each mouse intraperitoneally (IP).Mice were treated weekly at day 5, 12, 19 using indicated doses. Weeklyluminance body imaging was taken on day 10, 17, 24 for monitoring tumorvolume in each animal. At termination of the study, blood, spleen, boneand bone marrow were collected for histopathology study.

Results

mAb2-containing anti-CD38xanti-CD28xanti-CD3 trispecific bindingproteins with the IgG4 FALA variant Fc region caused markedly reducedlevels of non-specific IFN-γ release in human PBMCs, as compared withsimilar binding proteins with wild-type Fc regions (FIG. 18A). Similarresults were observed with release of IL-2 (FIG. 18B) and TNF-α (FIG.18C). Importantly, cytokine release was significantly lower for these Fcvariant trispecific binding proteins than for the benchmark bispecificanti-CD38xanti-CD3 bispecific antibody. These results imply improvedsafety profile of trispecific binding proteins with Fc variants due to areduction in non-specific cytokine release.

IgG1 and IgG4 Fc variants of both mAb2-containingCD38_(VH1)xCD28_(sup)xCD3_(mid) and mAb6-containingCD38_(HH71370)xCD28_(sup)xCD3_(mid) also led to in vitro activation ofhuman PBMCs (FIG. 18D).

Immune costimulation by ligation of CD28 is required for T cell survivaland proliferation; TCR signaling alone does not result in T cellproliferation (Sharmee and Allison (2015) Science 348:56-61).Trispecific binding protein CD38_(VH1)xCD28_(sup)xCD3_(mid) led toinduction of Bcl-xL in both CD4+ and CD8+ T cells, whereas removingeither the CD28 or the CD3 antigen binding domains eliminated thiseffect (FIGS. 19A&19B). These results demonstrate, as expected, thatboth CD3 and CD28 binding arms are required for Bcl-xL induction in Tcells by the anti-CD38 trispecific binding proteins, thereby deliveringa pro-survival signal to T cells. The anti-CD28 arm of the trispecificbinding protein is critical to upregulation of pro-survival Bcl-xL in Tcells. When compared with a benchmark anti-CD38xanti-CD3 bispecificantibody, CD38_(VH1)xCD28xCD3 trispecific binding protein led to greaterupregulation of Bcl-xL in CD4+ and CD8+ T cells (FIGS. 19C & 19D).

To determine the primary driver of T cell activation byCD38_(VH1)xCD28_(sup)xCD3_(mid) trispecific binding proteins, IL-2expression in a Jurkat T cell reporter Jurkat line containing human IL2promoter driving luciferase reporter was used to measure activation.Knockout mutation of the anti-CD3 binding domain led to elimination of Tcell activation, demonstrating that T cell activation is a specificeffect of CD3 ligation (FIG. 19E). These results show that anti-CD3 isthe primary driver of T cell activation by trispecific binding proteins,and that anti-CD28 also contributes significantly to the T cellactivation signal. The primary driver of cytokine release was alsoexamined using human PBMCs (FIG. 19F). By examining release of TNF,IFNg, IL-2, IL-6, and IL-10, it was found that CD3 is the primarydeterminant of cytokine release. CD28 was found to have the leastcontribution. Trispecific binding proteins with anti-CD38_(VH1) werealso found to induce less cytokine release than the benchmarkcomparator.

T cell proliferation was also examined. Activation of T cells usinganti-CD38xanti-CD28xanti-CD3 trispecific binding protein with IgG4 FALAvariant Fc led to greater proliferation than benchmark or isotypecontrol (FIG. 19G). This activation was reduced upon mutation of theanti-CD28 or anti-CD3 antigen binding domains (FIG. 20).

The humanized NSG mouse model was next used for in vivo solid tumorgrowth experiments. Mice were implanted at week 6-8 with RPMI8226 humanmyeloma cells. At week 9, human PBMCs were introduced by ip injection,hPBMCs were reconstituted, and anti-tumor therapy was administered atweeks 10-13. 56 PBMC humanized NSG female mice were implanted with 5million RPMI8226 cells in 50% matrigel, and mice were selected andrandomized into the study at day 5 or 6 post-implantation. This modelserves as an in vivo tumor model in mice with human mature T cells.

Trispecific binding proteins were also assayed in an NSG mouse model ofdisseminated tumor growth, using NCI-H929-Luc human myeloma cells andassaying tumor growth by bioluminescence.CD38_(VH1)xCD28_(sup)xCD3_(mid) trispecific binding protein with IgG4FALA Fc variant showed a statistically significant, dose-dependentanti-tumor efficacy in this disseminated tumor model at 30 μg/kg (FIG.21). CD38_(HHY1370)xCD28_(sup)xCD3_(mid) trispecific binding proteinwith IgG4 FALA Fc variant also showed statistically significant,dose-dependent anti-tumor efficacy in this disseminated tumor model at30 μg/kg and 10 μg/kg (FIG. 22).

Trispecific binding proteins mAb2-containingCD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 FALA and mAb6-containingCD38_(hhy1370)xCD28_(sup)xCD3_(mid) IgG4 FALA showed potent in vitrotumor killing activities against NCI-H929-Luc myeloma cells using humanPBMCs from two donor humanized NSG mice (FIGS. 23A & 23B). TheCD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 FALA trispecific binding proteinshowed superior in vivo tumor activity, as compared to benchmarkbispecific antibody, in the disseminated tumor model (FIG. 23C).P-values of comparison with vehicle control (PBS) were determined asfollows: CD38_(VH1)xCD28_(sup)xCD3_(mid) IgG4 FALA: p=0.0023;CD38_(HHY1370)xCD28_(sup)xCD3_(mid) IgG4 FALA: p=0.0088; benchmarkbispecific CD38xCD3 Fab-scFv-Fc: p=0.6045.

Taken together, these results demonstrate an improved anti-CD38candidate with variant Fc region that shows increased half-life, reducednonspecific cytokine release, and greater in vivo anti-tumor efficacy.

Example 9: Further Characterization of Anti-CD38 Trispecific BindingProteins

Two signals are required to stimulate T cells for optimal effectorfunction and sustained proliferation. Activation mediated by the T cellreceptor (TCR)-CD3 complex induces transcriptional activation leading tocytokine secretion. Engagement of a second surface membrane protein,CD28, stimulates an alternative signal transduction pathway and inhibitsprogrammed cell death (Esensten J H, Helou Y A, et al. Immunity44:973-88(2016); Hui E, Cheung J, et al. Science 355:1428-1433(2017)).In the absence of a second signal, T cell stimulation through CD3 alonetypically leads to activation-induced cell death (Chai J G, Lechler R I.Int Immunol. 9:935-44(1997)). It was hypothesized that the T cellproliferation could be increased by combining specificities againstthese two different targets.

FIG. 24A shows a luciferase reporter assay that was conducted usingGloResponse™ IL2-luc2P Jurkat Cells (Promega) after stimulation byCD38_(VH1)/CD28_(sup)xCD3_(mid) and its single binding site KO andtriple KO mutants at 10 nM concentration. These data illustrate thecontribution of CD28 to T cell stimulation (e.g., NFAT signaling).

Using the cross-over dual variable (CODV) bispecific Ab format(Steinmetz A, et al. MAbs 8: 867-878(2016)), combinations of α-CD3ε(signal 1) and α-CD28 (signal 2) Fv's were evaluated to determinewhether dual engagement of these cell surface molecules could stimulateand sustain T cell activation (FIG. 24B). A medium affinity (K_(D)˜20nM) anti-CD3ε Ab was used to avoid high affinity T cell receptorstimulation that causes high level cytokine release and increases therisk of cytokine release syndrome. This CD3 agonist was tested at bothpositions of the dual arm in combination with a superagonistic CD28 Abshown previously to support T cell proliferation (Waibler Z, Sender L Y,et al. PLoS One 3:e1708(2008)). These monovalent bispecific antibodieswere incubated with human PBMCs in vitro, and T cell stimulation wasmeasured by secretion of interferon-γ and IL-2. While both orientationswere active, optimal release of these cytokines was observed with CD3 inthe proximal and CD28 in the distal position (FIG. 24B). This dual armwas then selected for pairing with an anti-CD38 antibody in thetrispecific Ab format (Xu L, Pegu A, et al. Science 358:85-90 (2017)).

FIG. 25 shows that upregulation of Bcl-2 family member Bcl-xL in primaryT cells induced by CD38_(VH1)/CD28_(sup)xCD3_(mid) is CD28-dependent.These data illustrate the contribution of CD28 to T cell survival.

FIG. 26 shows that anti-CD28 in the trispecific Ab provided secondarysignaling essential for supporting primary T cell proliferation invitro. These data illustrate the contribution of CD28 to T cellproliferation.

Taken together, these data show that anti-CD28_(sup) in theCD38_(VH1)/CD28_(sup)xCD3_(mid) trispecific antibody providessignificant functionalities in T cell activation, survival andproliferation in vitro.

FIG. 27 shows the configuration of the trispecific antibody, coded byparental antibody. Also shown is a structure model of theCD38_(VH1)/CD28_(sup)xCD3_(mid) trispecific Ab obtained based on crystalstructures of anti-CD38 VH1 Fab and CD28_(sup)/CD3_(mid) CODV Fab(right).

FIGS. 28A & 28B show that multiple myeloma (MM) cells with high(RPMI-8226; FIG. 28A) and low (KMS-11; FIG. 28B) CD38 surface expressionwere lysed efficiently by human PBMCs (E:T=10:1) incubated with variousconcentrations of the trispecific Ab. Contribution to the killingactivity by each binding site was demonstrated by binding site KOmutations. These data demonstrate that the CD38 trispecific antibodylysed human MM cells through recognition of both CD38 and CD28. CD28expressed on Multiple Myeloma cells provided a secondary target by theCD38_(VH1)/CD28_(sup)xCD3_(mid) FALA trispecific antibody, significantlyenhancing its killing activity.

FIGS. 29A-29C show that the anti-CD28_(sup)KO mutant of the trispecificAb exhibited markedly reduced anti-tumor activity against CD38_(high),CD38_(mid) and CD38_(low) MM cells in vitro. Shown are assays usingRPMI-8226 (FIG. 29A), U266 (FIG. 29B), or KMS-11 (FIG. 29C) cells. CD28expression on MM cells increased their susceptibility to cytolysismediated by the CD38 trispecific antibody.

FIG. 30 shows the results of an in vivo efficacy study in a disseminatedhuman multiple myeloma cell line model using an NSG mouse reconstitutedwith in vitro amplified human primary T cells. Reduction of tumor burdenin CD38_(VH1)/CD28_(sup)xCD3_(mid)_FALA trispecific antibody treatmentgroups was dose-dependent and statistically different. Thus the CD38trispecific antibody conferred protection against disseminated human MMcell tumor growth in vivo.

A microscopic study further demonstrated cancer cell killing by primaryhuman T cells mediated by the trispecific antibody in vitro. FIGS. 31A &31B show microscopic images demonstrating RPMI-8226 (human MM cells,labeled with CellTracker Deep Red dye) lysed by human PBMCs mediated byCD38_(VH1)/CD28_(sup)xCD3_(mid_)FALA trispecific antibody. FIG. 31Ashows control, while FIG. 31B shows lysis mediated byCD38_(VH1)/CD28_(sup)xCD3_(mid_)FALA trispecific antibody. 10:1 E:Tratio was used with 24 hr. incubation. Exposure to the triple nullmutant control Ab induced minimal changes in tumor cell viability over24 hours (FIG. 31A). In contrast, incubation with the active CD38trispecific Ab induced substantial clustering of T cells around tumorcells, leading to their nearly complete lysis (FIG. 31B). These datademonstrate that CD38/CD3xCD28 trispecific antibody mediated cytolysisof myeloma cells by T cells.

Example 10: Modification of Fc Receptor Binding Sites in the IgG4 FcRegion of Anti-CD38 Trispecific Binding Proteins Reduces Non-SpecificInflammation

The Fc region of antibodies binds to cellular receptors on monocytes andNK cells that induce non-specific inflammation, which could predisposetreatment subjects to cytokine release syndrome. The role of Fcreceptors in stimulating cytokine release was examined by testingmutants that eliminated FcγI, FcγIIa,b and FcγIII binding. For thispurpose, an IgG4 isotype that does not fix complement was used.

Binding in the surface plasmon resonance (SPR) assay using the BIAcoresystem (Pharmacia Biosensor; Piscataway, N.J.) was used to measure theaffinity of the specified IgG4 Fc variants to indicated human Fcreceptors immobilized on the chip. IgG4 Fc variants were used at 150 nM.The binding to human FcRn was carried using ELISA by coating the humanFcRn antigen on the plate.

A mutation (“FALA”) in the Fc region that has been previously described(Alegre M L, Peterson L J, et al. Transplantation 57:1537-43(1994); seealso Examples 7-9) eliminated binding to all Fc receptors (FIGS. 32 &33). When tested for non-specific cytokine release, it abrogated IFN-γrelease in unstimulated PBMC while allowing for maximal stimulation inthe presence of tumor targets that expressed CD38 (FIG. 34, left vs.right. FALA). This Fc mutant retained its cytolytic activity on myelomatumor targets comparable to Fc wild type control in cells expressingdifferent levels of CD38 (FIG. 35). Similarly, another mutated IgG4 Fcregion (“PVA,” see FIG. 32) that eliminated binding to Fc receptors(FIG. 33) also abrogated IFN-γ release in unstimulated PBMC whileallowing for maximal stimulation in the presence of tumor targets thatexpressed CD38 (FIG. 34, left vs. right, PVA) and retained its cytolyticactivity on myeloma tumor targets comparable to Fc wild type control incells expressing different levels of CD38 (FIG. 35).

Example 11: Contribution of Anti-CD38 and Anti-CD28 to CytolyticActivity of Trispecific Binding Proteins

Trispecific Ab mutants for each binding site or combination of bindingsites were generated and tested for their cytolytic activity againstmultiple myeloma cell lines with high (RPMI-8226) or low (KMS-11) CD38and CD28 surface expression.

As shown above in FIGS. 28A & 28B, mutation of CD3 specific bindingabrogated cytolysis, while anti-CD38 and CD28 null mutations showedmarkedly reduced killing, but some residual function was retained,indicating that both anti-CD38 and CD28 contributed to tumor cellkilling.

Compared to daratumumab (the α-CD38 monoclonal antibody approved for thetreatment of multiple myeloma; see McKeage K. Drugs. 76:275-81(2016)),the trispecific Ab showed a remarkable 3-4 log higher killing potency invitro against different CD38 expressing cell lines, including bothCD38_(high) and CD38_(low) multiple myeloma cells (FIG. 36).

Example 12: CD38/CD3xCD28 Ab Stimulates Central Memory CD4 and CD8, Th1and Antigen-Specific Responses

To determine whether the CD38/CD3xCD28 trispecific Ab could enhancecellular immune function, the phenotype of expanded T cells in vitro wasevaluated.

Materials and Methods

Peripheral blood mononuclear cells were isolated from blood of healthyhuman donors collected by Research Blood Components, LLC (Boston,Mass.). The PBMC were added to antibody-coated plates (350 ng/well)(5×10⁵ cells/mL), as previously described above, and incubated at 37° C.for 3 and 7 days. The cells were collected at specific time points andanalyzed by flow cytometry for T cell subsets: naïve (CCR7+ CD45RO−),Tcm (CCR7+ CD45RO+), Tem (CCR7− CD45RO+), Tregs (CD4+ Foxp3+ CD25hi).Cells were also treated with monensin (GolgiStop) (BD Biosciences, CA)for at least 6 hours before flow staining to determine intracellularcytokine expression: Th1 (CD4+ IFN-γ+), Th2 (CD4+ IL-4+), and Th17 (CD4+IL-17+). CMV pp65-specific CD8+ T cells were detected usingfluorescent-conjugated pentamer restricted to the PBMC donors' HLA(A*02:01/NLVPMVATV) (ProImmune, Oxford, UK). PBMC was obtained fromHemaCare (Van Nuys, CA) from donors with known CMV positive populationsand HLA types. PMBC from donors negative for the restricting HLA typewas used as negative control. Staining was done as per manufacturer'sprotocol.

Results

Human PBMCs were incubated for 7 days with the trispecific Ab or atriple mutant negative control in the absence of cytokines. Analysis ofthe CD4 subsets revealed the greatest proliferation in the centralmemory pool, with a smaller increase in effector memory cells (FIG.37A). Analysis of the CD4 subset also revealed the greatestproliferation of Th1 cells (>6-fold) compared to Th2 or Th17 cells (FIG.37B). In the CD8 subset, there was a >150-fold increase in the centralmemory CD8 subset by day 7, with a lesser increase in effector memorycells (FIG. 37C). Importantly, pre-existing antigen-specific CD8responses to CMV, directed to the pp65 epitope in seropositive HLA-A2donors using tetramer staining (Gratama J W, van Esser J W, et al. Blood98:1358-1364(2001)), increased >44-fold in the presence of the CD38trispecific compared to negative control (FIG. 37D).

Taken together, these data indicate that the CD38 trispecific Abstimulates Th1 function and protective CD8 memory T cell responses thatare likely to enhance anti-tumor immunity in vivo.

Example 13: CD28 on Multiple Myeloma Cells Increases T Cell Recognitionand Cytolysis

Materials and Methods

Flow Cytometry

Levels of CD28 and CD38 on the indicated tumor cell lines weredetermined by flow cytometry using QIFI kit (Dako, Denmark, cat.#K007811) as described (S4). Briefly, mouse anti-human CD38 (cloneAT13/5, mouse IgG1, Santa Cruz Biotech, cat #59028) and anti-human CD28(clone CD28.2, mouse IgG1, k, BioLegend, cat #3029123) were used asprimary antibodies (at 10 ug/ml) for detecting surface CD38 and CD28 oncell lines using manufacturer's protocol. Surface densities werecalculated using QIFI kit calibration standards and formulation providedby the kit.

Generation of CD28 KO Human Multiple Myeloma Cell Lines UsingCRISPR-Mediated Knockout

For KMS-11 cell line, knockout of CD28 was performed using the CRISPRCD28 human knockout kit (Origene). The kit contains a donor vector witha GFP-puromycin functional cassette and two distinct Cas-9 vectors eachwith a different predesigned guide RNA (gRNA 1: TACCTGTTACTTGAATTGAA(SEQ ID NO:117) and gRNA 2: ATTTTTTGAGGTCTTCCAAT (SEQ ID NO:118)targeting CD28 Exon 1 and Intron 1, respectively). Cells were seeded ina 6-well plate at a density of 3·10⁵ cell/well in RPMI 1640 GlutaMAXMedium supplemented with 10% FBS and co-transfected after 24 hours withall three vectors using Lipofectamine™ 3000 Transfection Reagent(ThermoFisher Scientific) according to the manufacturer's instructions.Puromycin was added 48 hours after the transfection at a finalconcentration of 0.5 μg/mL. Transfection efficiency was 30% when using6.25 μL/well of Lipofectamine™ 3000, 1.25 μg of each Cas-9/gRNA vectorand 2.5 jig of the GFP-puromycin donor vector. Knockout of CD28 on RPMI8226 cell line was performed using the CD28 sgRNA CRISPR All-in-OneLentivirus set (Applied Biological Materials Inc.). It consists of a setof 3 lentiviral all-in-one vectors expressing the Cas9 gene, a Puromycinresistance gene and a different sgRNA (sgRNA 1: ATTGTCGTACGCTACAAGCA(SEQ ID NO:119) targeting CD28 exon 2 and sgRNA 2: CAAAAGGGCTTAGAAGGTCC(SEQ ID NO:120) and sgRNA 3: CTATAGCTTGCTAGTAACAG (SEQ ID NO:121)targeting CD28 exon 3). Cells were infected using a spinoculationprotocol with 2·10⁵ cells mixed with lentiviral particles (10 UI/cell),8 μg/mL of polybrene and 1:100 ViralPlus Transduction Enhancer (AppliedBiological Materials Inc.). The mix was pre-incubated for 20 minutes atroom temperature, then centrifuged for 30 minutes at 32° C. and 800×gand seeded in a 12-well plate. Puromycin was added 48 hours after theinfection at a final concentration of 0.25 μg/mL. Both cells lines werepassaged for at least 5 times (or until cell viability becomes stable)before confirming CD28 knockdown at the protein level by flow cytometry(CD28 PE Clone 28.2 antibody-BioLegend). CD28 negative cells were thensorted on a Sony SH800S Cell Sorter and cultured in media supplementedwith 0.25 μg/mL or 0.5 μg/mL puromycin, 100 Unit/mL penicillin and 100μg/mL streptomycin (ThermoFisher Scientific). Cells were then cloned bylimiting dilution and CD28 knockout validated by flow cytometry and byPCR and Sanger sequencing using the forward and reverse primers listedbelow.

TABLE R List of forward and reverse primers. Ampli- Cell Ampli- con linecon Primers size Comments KMS- CD28_ F: 1475  Validates the  11 GFP_CACAACCTGTCC bp insertion of left CCATCCTATGAA the GFP-puromycin(SEQ ID NO:  cassette 122) (left side) R: AAGCTGCCATCC AGATCGTTATCG(SEQ ID NO:  123) CD28_ F: 1233  Validates the  PURO_ CCAAATTAAGGG bpinsertion of right CCAGCTCATTCC the GFP-puromycin (SEQ ID NO:  cassette124) (right side) R: ACCTGTACATCC TTGGGCAAATCC (SEQ ID NO:  125) CD28_F: Vari- Region surrounding Exon_ GTCAGGATGCCT able; CD28 exon1-intron11 TGTGGTTTGAGT WT =  junction  (SEQ ID NO:  720 corresponding to 126)the insertion R: site of the CAGAGCTTCCAG GFP-puromycin  AGCCAATCTACcassette (SEQ ID NO:  and/or indels 127) following CRISPR cleavage RPMIExon_ F: Vari- Region surrounding 8226 2 CCATGTACTGCC able; CD28 exon 2TTCTGGGTGAAA WT = corresponding to  (SEQ ID NO:  682 CRISPR 128)cleavage sites R: CCACTGACCACA bp ACCCAGTTTT (SEQ ID NO:  129) Exon_ F:Vari- Region surrounding 3 AGGCCATTGGAA able; CD28 exon 3 GTCACCGTTTcorresponding to (SEQ ID NO:  WT = CRISPR 130) 816 cleavage sites R:GCCAACATTGTC bp CATTGGCTTCAG (SEQ ID NO:  131)

Results

CD28 was incorporated into the trispecific Ab to improve T cellproliferation and survival; however, when cell surface markers wereevaluated on multiple myeloma cells, a majority of cell lines (>95%)were found to express the CD28 glycoprotein (Table S).

TABLE S Cell surface expression of CD28 and CD38 on human multiplemyeloma cell lines. Cell line CD28 CD38 JJN3 1,040 50 KMS-11 (p17)35,345 2,845 U266 170,525 13,825 L-363 9,145 17,500 MM.1S 56,560 44,535MM.1R 66,220 49,160 KMS-12BM 0 62,845 RPMI 8226 83,915 136,255 NCI-H92922,065 153,620 LP-1 7,420 372,835 U266-CD38++ 126,465 633,805 JJN3-CD380 714,805 MOLP-8 245 885,030 NCI-H929-CD38++ 22,935 981,310 RPMI8226-CD38++ 86,320 1,419,120

It has been reported that CD28 expression, while absent on normal plasmacells (Almeida J, et al. British J. of Haematol. 107:121-131(1999)), canbe detected on primary myeloma plasma cells of in approximately onethird of newly diagnosed patients (Mateo G, et al. Clin. Cancer Res.11:3661-3667(2005)). Furthermore, it increases in frequency duringmyeloma progression and correlates with poor prognosis and aggressivefeatures of myeloma (Robillard N, Jego G, et al. Clin Cancer Res.4:1521-6(1998); Nair J R, Carlson L M, et al. J Immunol.187:1243-53(2011)).

To determine whether CD28 expression on target cells could improve Tcell recognition and lysis, the activity of wild type vs. CD28 nulltrispecific Abs on three independent myeloma lines with varying degreesof CD38 and CD28 expression was compared. As noted above in Example 9andshown in FIGS. 29A-29C, cytolysis was observed on all three lines,regardless of CD28 expression level by the CD38 trispecific Ab; however,against all cell lines, cytolytic activity of the CD28 null trispecificAb was reduced by 30-100 fold, with the most substantial decrease in theKMS-11 cell line that showed lowest CD38 expression (cf. WT vs. ΔCD28,FIG. 29C vs. FIG. 29A or FIG. 29B).

The contribution of CD28 to tumor cell recognition and lysis was furtherconfirmed using CRISPR-mediated knockout of CD28 on the myeloma targetcell. CD28 expression was undetectable on CD28KO KMS-11 cells comparedto the parental line (FIG. 38, upper panel, right vs. left). Thesensitivity of the CD28KO KMS-11 cells to T cell cytolysis wasconcomitantly reduced 10-100 fold (FIG. 38, lower panel, left).Consistent with this effect, no difference in cytolysis was seen withthe CD38 trispecific compared to the CD28 null mutant trispecific on theCD38 KO target cells (FIG. 38, lower panel, right).

Example 14: Cytolysis of the CD38 Trispecific Ab Against CD38⁺Hematological Cancer Cell Lines

The previous Examples demonstrate trispecific anti-CD38/CD3/CD28antibodies that promote cytolysis of multiple myeloma cells. The abilityof these antibodies to promote cytolysis of other cancer cell lines wastested.

As shown in FIG. 39, CD38/CD28xCD3 trispecific FALA mutant Ab hadcytolytic activity targeting CD38⁺CD28⁻ lines, including acutemyelocytic leukemia (AML (KG-1)), a B cell lymphoma (OCI-Ly19), acute Tlymphocytic leukemia (ALL (KOPN8)), and chronic lymphocytic lymphoma(CLL(Z-138)). This demonstrates that trispecific anti-CD38/CD3/CD28antibodies have activity against other CD38+ hematological cancer celllines, including those that are CD28−.

Example 15: In Vitro Activation of Human PBMC by α-CD28 SuperagonistRequires Bivalency of the Antibody

In vitro activation of human PBMC by TGN1412 (TGN), single binding armTGN1412(TGNslg) and single binding arm anti-CD28_(sup)(CD28_(sup)slg)was performed as described (Findlay L, Eastwood D, et al. J ImmunolMethods 352:1-12 (2010)), 10⁵ human PBMCs were seeded onto polypropylene96-well plates dry coated with indicated antibodies (1 ug/well) for 24hours as previously described. Cytokines such as IFN-γ, TNF-α, and IL2in the supernatant were measured by Luminex as described in Example 8.

The inclusion of a single specificity of the CD28 superagonist alsoreduced non-specific cytokine release seen with the CD28 superagonistmAb (FIG. 40) previously associated with its adverse effects as a nativeIgG in humans (Suntharalingam G, Perry M R., et al. N Engl J Med355:1018-1028(2006)). Instead, co-stimulation by the trispecific Abincreased the potency and survival of T cells that lyse and protectagainst tumors. More interestingly, the CD38 trispecific Abpreferentially amplify Th1 and Tcm cell populations in vitro, exhibitingan important feature that differs from α-CD3 agonist or α-CD28superagonist monoclonal Abs used to propagate Treg to induce tolerance(Penaranda Cl, Tang Q, Bluestone J A. J Immunol. 187:2015-22. (2011);Tabares P, Berr S. et al. Eur J Immunol. 44:1225-36(2014)).

Taken together, the data presented in Examples 1-15 demonstratetrispecific anti-CD38/CD3/CD28 antibodies that stimulate potentanti-tumor immunity by engaging two T cell signaling receptors andenhancing target cell recognition. CD3 and CD28 binding triggered T cellreceptor signaling that upregulated Bcl-xl and inhibited T cellapoptosis, increasing T cell effector function and survival. The thirdarm of the trispecific Ab interacted with CD38, which is highlyexpressed on multiple myeloma cells. Coincidentally, CD28 is alsoexpressed on cells from most myelomas; therefore the trispecific Abimproved targeting at the same time it enhanced T cell activation andcytolysis. The optimized trispecific antibody lysed myelomacells >1000-fold more effectively than daratumumab, an anti-CD38 mAbtherapeutic, and showed potent in vivo anti-tumor activity in ahumanized mouse model.

While the disclosure includes various embodiments, it is understood thatvariations and modifications will occur to those skilled in the art.Therefore, it is intended that the appended claims cover all suchequivalent variations that come within the scope of the disclosure. Inaddition, the section headings used herein are for organizationalpurposes only and are not to be construed as limiting the subject matterdescribed.

Each embodiment herein described may be combined with any otherembodiment or embodiments unless clearly indicated to the contrary. Inparticular, any feature or embodiment indicated as being preferred oradvantageous may be combined with any other feature or features orembodiment or embodiments indicated as being preferred or advantageous,unless clearly indicated to the contrary.

All references cited in this application are expressly incorporated byreference herein.

What is claimed is:
 1. A binding protein comprising an antigen bindingsite that binds a CD38 polypeptide, wherein the antigen binding sitecomprises: (a) an antibody heavy chain variable (VH) domain comprising aCDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ IDNO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising theamino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ IDNO:38), and a CDR-H3 sequence comprising the amino acid sequence ofARTGGLRRAYFTY (SEQ ID NO:33); and (b) an antibody light chain variable(VL) domain comprising a CDR-L1 sequence comprising the amino acidsequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), aCDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35)or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acidsequence of QQNKEDPWT (SEQ ID NO:36).
 2. The binding protein of claim 1,wherein the antigen binding site comprises: (a) an antibody heavy chainvariable (VH) domain comprising a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprisingthe amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ IDNO:33); and (b) an antibody light chain variable (VL) domain comprisinga CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS(SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36).
 3. The binding protein of claim 1 or claim2, wherein the VH domain comprises the sequence, from N-terminus toC-terminus, FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; wherein FR1 comprisesthe sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86),QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87), or QVQLVQSGAEVVKPGASVKMSCKAS(SEQ ID NO:88); wherein FR2 comprises the sequence MHWVKEAPGQRLEWIGY(SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO:91); wherein FR3comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ IDNO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); andwherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:96).
 4. Thebinding protein of claim 1 or claim 2, wherein the VH domain comprisesthe amino acid sequence of SEQ ID NO:5, and the VL domain comprises theamino acid sequence of SEQ ID NO:6.
 5. The binding protein of claim 4,wherein the binding protein comprises an antibody heavy chain comprisingthe amino acid sequence of SEQ ID NO:7 and an antibody light chaincomprising the amino acid sequence of SEQ ID NO:8.
 6. The bindingprotein of claim 1 or claim 2, wherein the VH domain comprises the aminoacid sequence of SEQ ID NO:17, and the VL domain comprises the aminoacid sequence of SEQ ID NO:18.
 7. The binding protein of claim 6,wherein the binding protein comprises an antibody heavy chain comprisingthe amino acid sequence of SEQ ID NO:19 and an antibody light chaincomprising the amino acid sequence of SEQ ID NO:20.
 8. The bindingprotein of claim 1 or claim 2, wherein the VH domain comprises the aminoacid sequence of SEQ ID NO:21, and the VL domain comprises the aminoacid sequence of SEQ ID NO:18.
 9. The binding protein of claim 8,wherein the binding protein comprises an antibody heavy chain comprisingthe amino acid sequence of SEQ ID NO:22 and an antibody light chaincomprising the amino acid sequence of SEQ ID NO:20.
 10. The bindingprotein of claim 1 or claim 2, wherein the VH domain comprises the aminoacid sequence of SEQ ID NO:23, and the VL domain comprises the aminoacid sequence of SEQ ID NO:18.
 11. The binding protein of claim 10,wherein the binding protein comprises an antibody heavy chain comprisingthe amino acid sequence of SEQ ID NO:24 and an antibody light chaincomprising the amino acid sequence of SEQ ID NO:20.
 12. The bindingprotein of claim 1, wherein the antigen binding site comprises: (a) anantibody heavy chain variable (VH) domain comprising a CDR-H1 sequencecomprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38),and a CDR-H3 sequence comprising the amino acid sequence ofARTGGLRRAYFTY (SEQ ID NO:33); and (b) an antibody light chain variable(VL) domain comprising a CDR-L1 sequence comprising the amino acidsequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising theamino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequencecomprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).
 13. Thebinding protein of claim 1 or claim 12, wherein the VH domain comprisesthe sequence, from N-terminus to C-terminus,FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; wherein FR1 comprises the sequenceQVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQID NO:87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); wherein FR2comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) orMHWVKEAPGQGLEWIGY (SEQ ID NO:91); wherein FR3 comprises the sequenceNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) orNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and wherein FR4comprises the sequence WGQGTLVTVSS (SEQ ID NO:96).
 14. The bindingprotein of claim 1 or claim 12, wherein the VH domain comprises theamino acid sequence of SEQ ID NO:13, and the VL domain comprises theamino acid sequence of SEQ ID NO:14.
 15. The binding protein of claim14, wherein the binding protein comprises an antibody heavy chaincomprising the amino acid sequence of SEQ ID NO:15 and an antibody lightchain comprising the amino acid sequence of SEQ ID NO:16.
 16. A bindingprotein comprising an antigen binding site that binds a CD38polypeptide, wherein the antigen binding site comprises: (a) an antibodyheavy chain variable (VH) domain comprising a CDR-H1 sequence comprisingthe amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequencecomprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and aCDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ IDNO:43); and (b) an antibody light chain variable (VL) domain comprisinga CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ IDNO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQID NO:45), and a CDR-L3 sequence comprising the amino acid sequence ofLQDYIYYPT (SEQ ID NO:46).
 17. The binding protein of claim 16, whereinthe VH domain comprises the amino acid sequence of SEQ ID NO:9, and theVL domain comprises the amino acid sequence of SEQ ID NO:10.
 18. Thebinding protein of claim 16 or claim 17, wherein the binding proteincomprises an antibody heavy chain comprising the amino acid sequence ofSEQ ID NO:11 and an antibody light chain comprising the amino acidsequence of SEQ ID NO:12.
 19. The binding protein of any one of claims1-18, wherein the antigen binding site cross-reacts with anextracellular domain of a human CD38 polypeptide and an extracellulardomain of a cynomolgus monkey CD38 polypeptide.
 20. The binding proteinof claim 19, wherein the antigen binding site binds a human CD38polypeptide comprising the amino acid sequence of SEQ ID NO:1.
 21. Thebinding protein of claim 20, wherein the antigen binding site binds thehuman CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1with an equilibrium dissociation constant (K_(D)) of 2.1 nM or less. 22.The binding protein of claim 19, wherein the antigen binding site bindsa human isoform E CD38 polypeptide comprising the amino acid sequence ofSEQ ID NO:105.
 23. The binding protein of any one of claims 19-22,wherein the antigen binding site binds a cynomolgus monkey CD38polypeptide comprising the amino acid sequence of SEQ ID NO:30.
 24. Thebinding protein of claim 23, wherein the antigen binding site binds thecynomolgus monkey CD38 polypeptide comprising the amino acid sequence ofSEQ ID NO:30 with an equilibrium dissociation constant (K_(D)) of 1.3 nMor less.
 25. The binding protein of any one of claims 1-24, wherein thebinding protein is a chimeric or humanized antibody.
 26. The bindingprotein of any one of claims 16-24, wherein the binding protein is ahuman antibody.
 27. The binding protein of any one of claims 1-24,wherein the binding protein is a monoclonal antibody.
 28. The bindingprotein of any one of claims 1-24, wherein the binding protein comprisesone or more full-length antibody heavy chains comprising an Fc region.29. The binding protein of claim 28, wherein the Fc region is a human Fcregion comprising one or more mutations that reduce or eliminate Fcreceptor binding and/or effector function of the Fc region.
 30. Thebinding protein of claim 28, wherein the Fc region is a human IgG1 Fcregion.
 31. The binding protein of claim 30, wherein the human IgG1 Fcregion comprises amino acid substitutions at positions corresponding topositions 234, 235, and 329 of human IgG1 according to EU Index, whereinthe amino acid substitutions are L234A, L235A, and P329A.
 32. Thebinding protein of claim 30, wherein the human IgG1 Fc region comprisesamino acid substitutions at positions corresponding to positions 298,299, and 300 of human IgG1 according to EU Index, wherein the amino acidsubstitutions are S298N, T299A, and Y300S.
 33. The binding protein ofclaim 28, wherein the Fc region is a human IgG4 Fc region.
 34. Thebinding protein of claim 33, wherein the human IgG4 Fc region comprisesamino acid substitutions at positions corresponding to positions 228 and409 of human IgG4 according to EU Index, wherein the amino acidsubstitutions are S228P and R409K.
 35. The binding protein of claim 33or claim 34, wherein the human IgG4 Fc region comprises amino acidsubstitutions at positions corresponding to positions 234 and 235 ofhuman IgG4 according to EU Index, wherein the amino acid substitutionsare F234A and L235A.
 36. The binding protein of claim 33 or claim 34,wherein the human IgG4 Fc region comprises amino acid substitutions atpositions corresponding to positions 233-236 of human IgG4 according toEU Index, wherein the amino acid substitutions are E233P, F234V, L235A,and a deletion at
 236. 37. The binding protein of any one of claims1-24, wherein the binding protein comprises an antibody F(ab), F(ab′)2,Fab′-SH, Fv, or scFv fragment.
 38. The binding protein of any one ofclaims 1-37, wherein the binding protein is conjugated to a cytotoxicagent or label.
 39. The binding protein of any one of claims 1-37,wherein the binding protein is a bispecific binding protein comprisingthe first antigen binding site that binds the CD38 polypeptide and asecond antigen binding site.
 40. The binding protein of any one ofclaims 1-37, wherein the binding protein is a trispecific bindingprotein comprising the first antigen binding site that binds the CD38polypeptide, a second antigen binding site, and a third antigen bindingsite.
 41. The binding protein of claim 40, wherein the first antigenbinding site binds the extracellular domain of a human CD38 polypeptide,and wherein the second and third antigen binding sites each bind aT-cell surface protein.
 42. The binding protein of claim 41, wherein thefirst antigen binding site binds the extracellular domain of a humanCD38 polypeptide, and wherein (a) the second antigen binding site bindsa human CD28 polypeptide, and the third antigen binding site binds ahuman CD3 polypeptide, or (b) the second antigen binding site binds ahuman CD3 polypeptide, and the third antigen binding site binds a humanCD28 polypeptide.
 43. A binding protein comprising three antigen bindingsites that each bind one or more target proteins, wherein at least oneof the three antigen binding sites cross-reacts with an extracellulardomain of a human CD38 polypeptide and an extracellular domain of acynomolgus monkey CD38 polypeptide.
 44. The binding protein of claim 43,wherein the binding protein cross-reacts with a human CD38 polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:105. 45.The binding protein of claim 43 or claim 44, wherein the binding proteincross-reacts with a cynomolgus monkey CD38 polypeptide comprising theamino acid sequence of SEQ ID NO:30.
 46. The binding protein of any oneof claims 43-45, wherein the binding protein comprises an antigenbinding site that cross-reacts with an extracellular domain of a humanCD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38polypeptide and two antigen binding sites that each bind a T-cellsurface protein.
 47. The binding protein of claim 46, wherein thebinding protein comprises an antigen binding site that cross-reacts withan extracellular domain of a human CD38 polypeptide and an extracellulardomain of a cynomolgus monkey CD38 polypeptide, an antigen binding sitethat binds a human CD28 polypeptide, and an antigen binding site thatbinds a human CD3 polypeptide.
 48. The binding protein of any one ofclaims 43-47, wherein the binding protein comprises four polypeptidechains that form the three antigen binding sites, wherein a firstpolypeptide chain comprises a structure represented by the formula:V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I] and a second polypeptide chain comprisesa structure represented by the formula:V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3)  [II] and a thirdpolypeptide chain comprises a structure represented by the formula:V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3)  [III] and a fourth polypeptide chaincomprises a structure represented by the formula:V_(L3)-C_(L)  [IV] wherein: V_(L1) is a first immunoglobulin light chainvariable domain; V_(L2) is a second immunoglobulin light chain variabledomain; V_(L3) is a third immunoglobulin light chain variable domain;V_(H1) is a first immunoglobulin heavy chain variable domain; V_(H2) isa second immunoglobulin heavy chain variable domain; V_(H3) is a thirdimmunoglobulin heavy chain variable domain; C_(L) is an immunoglobulinlight chain constant domain; C_(H1) is an immunoglobulin C_(H1) heavychain constant domain; C_(H2) is an immunoglobulin C_(H2) heavy chainconstant domain; C_(H3) is an immunoglobulin C_(H3) heavy chain constantdomain; hinge is an immunoglobulin hinge region connecting the C_(H1)and C_(H2) domains; and L₁, L₂, L₃ and L₄ are amino acid linkers;wherein the polypeptide of formula I and the polypeptide of formula IIform a cross-over light chain-heavy chain pair, and wherein: (a) theV_(H1) domain comprises a CDR-H1 sequence comprising the amino acidsequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32)or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the aminoacid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L1) domaincomprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36); (b) the V_(H2) domain comprises a CDR-H1sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31)or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and aCDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQID NO:33), and the V_(L2) domain comprises a CDR-L1 sequence comprisingthe amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS(SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprisingthe amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or (c) the V_(H3)domain comprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) orIYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acidsequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3) domaincomprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS(SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequenceof QQNKEDPWT (SEQ ID NO:36).
 49. The binding protein of claim 48,wherein (a) the V_(H1) domain comprises a CDR-H1 sequence comprising theamino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequencecomprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and aCDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQID NO:33), and the V_(L1) domain comprises a CDR-L1 sequence comprisingthe amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequencecomprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);(b) the V_(H1) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprisingthe amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ IDNO:33), and the V_(L1) domain comprises a CDR-L1 sequence comprising theamino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequencecomprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);(c) the V_(H2) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprisingthe amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ IDNO:33), and the V_(L2) domain comprises a CDR-L1 sequence comprising theamino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequencecomprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);(d) the V_(H2) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprisingthe amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ IDNO:33), and the V_(L2) domain comprises a CDR-L1 sequence comprising theamino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequencecomprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);(e) the V_(H3) domain comprises a CDR-H1 sequence comprising the aminoacid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprisingthe amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ IDNO:33), and the V_(L3) domain comprises a CDR-L1 sequence comprising theamino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequencecomprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);or (h) the V_(H3) domain comprises a CDR-H1 sequence comprising theamino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequencecomprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and aCDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQID NO:33), and the V_(L3) domain comprises a CDR-L1 sequence comprisingthe amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequencecomprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).50. The binding protein of claim 49, wherein (a) the V_(H3) domaincomprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acidsequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acidsequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36); or (b) the V_(H3)domain comprises a CDR-H1 sequence comprising the amino acid sequence ofGYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acidsequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprisingthe amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_(L3)domain comprises a CDR-L1 sequence comprising the amino acid sequence ofQSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acidsequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising theamino acid sequence of QQNKEDPWT (SEQ ID NO:36).
 51. The binding proteinof claim 50, wherein (a) the V_(H3) domain comprises the amino acidsequence of SEQ ID NO:5, and the V_(L3) domain comprises the amino acidsequence of SEQ ID NO:6; (b) the V_(H3) domain comprises the amino acidsequence of SEQ ID NO:17, and the V_(L3) domain comprises the amino acidsequence of SEQ ID NO:18; (c) the V_(H3) domain comprises the amino acidsequence of SEQ ID NO:21, and the V_(L3) domain comprises the amino acidsequence of SEQ ID NO:18; (d) the V_(H3) domain comprises the amino acidsequence of SEQ ID NO:23, and the V_(L3) domain comprises the amino acidsequence of SEQ ID NO:18; or (e) the V_(H3) domain comprises the aminoacid sequence of SEQ ID NO:13, and the V_(L3) domain comprises the aminoacid sequence of SEQ ID NO:14.
 52. The binding protein of any one ofclaims 43-47, wherein the binding protein comprises four polypeptidechains that form the three antigen binding sites, wherein a firstpolypeptide chain comprises a structure represented by the formula:V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I] and a second polypeptide chain comprisesa structure represented by the formula:V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3)  [II] and a thirdpolypeptide chain comprises a structure represented by the formula:V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3)  [III] and a fourth polypeptide chaincomprises a structure represented by the formula:V_(L3)-C_(L)  [IV] wherein: V_(L1) is a first immunoglobulin light chainvariable domain; V_(L2) is a second immunoglobulin light chain variabledomain; V_(L3) is a third immunoglobulin light chain variable domain;V_(H1) is a first immunoglobulin heavy chain variable domain; V_(H2) isa second immunoglobulin heavy chain variable domain; V_(H3) is a thirdimmunoglobulin heavy chain variable domain; C_(L) is an immunoglobulinlight chain constant domain; C_(H1) is an immunoglobulin C_(H1) heavychain constant domain; C_(H2) is an immunoglobulin C_(H2) heavy chainconstant domain; C_(H3) is an immunoglobulin C_(H3) heavy chain constantdomain; hinge is an immunoglobulin hinge region connecting the C_(H1)and C_(H2) domains; and L₁, L₂, L₃ and L₄ are amino acid linkers;wherein the polypeptide of formula I and the polypeptide of formula IIform a cross-over light chain-heavy chain pair, and wherein: (a) theV_(H1) domain comprises a CDR-H1 sequence comprising the amino acidsequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising theamino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequencecomprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and theV_(L1) domain comprises a CDR-L1 sequence comprising the amino acidsequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising theamino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequencecomprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46); (b) theV_(H2) domain comprises a CDR-H1 sequence comprising the amino acidsequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising theamino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequencecomprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and theV_(L2) domain comprises a CDR-L1 sequence comprising the amino acidsequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising theamino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequencecomprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46); or (c)the V_(H3) domain comprises a CDR-H1 sequence comprising the amino acidsequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising theamino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequencecomprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and theV_(L3) domain comprises a CDR-L1 sequence comprising the amino acidsequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising theamino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequencecomprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46).
 53. Thebinding protein of claim 52, wherein the V_(H3) domain comprises aCDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ IDNO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK(SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequenceof ARMFRGAFDY (SEQ ID NO:43), and the V_(L3) domain comprises a CDR-L1sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), aCDR-L2 sequence comprising the amino acid sequence of AAS (SEQ IDNO:45), and a CDR-L3 sequence comprising the amino acid sequence ofLQDYIYYPT (SEQ ID NO:46).
 54. The binding protein of claim 53, whereinthe V_(H3) domain comprises the amino acid sequence of SEQ ID NO:9, andthe V_(L3) domain comprises the amino acid sequence of SEQ ID NO:10. 55.The binding protein of any one of claims 48-54, wherein (a) the V_(H1)domain comprises the amino acid sequence of SEQ ID NO:49, the V_(L1)domain comprises the amino acid sequence of SEQ ID NO:50, the V_(H2)domain comprises the amino acid sequence of SEQ ID NO:53, and the V_(L2)domain comprises the amino acid sequence of SEQ ID NO:54; (b) the V_(H2)domain comprises the amino acid sequence of SEQ ID NO:49, theV_(L2)domain comprises the amino acid sequence of SEQ ID NO:50, theV_(H1) domain comprises the amino acid sequence of SEQ ID NO:53, and theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:54; (c) theV_(H1) domain comprises the amino acid sequence of SEQ ID NO:51, theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:52, theV_(H2) domain comprises the amino acid sequence of SEQ ID NO:53, and theV_(L2) domain comprises the amino acid sequence of SEQ ID NO:54; (d) theV_(H2) domain comprises the amino acid sequence of SEQ ID NO:51, theV_(L2) domain comprises the amino acid sequence of SEQ ID NO:52, theV_(H1) domain comprises the amino acid sequence of SEQ ID NO:53, and theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:54 (e) theV_(H1) domain comprises the amino acid sequence of SEQ ID NO:49, theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:50, theV_(H2) domain comprises the amino acid sequence of SEQ ID NO:84, and theV_(L2) domain comprises the amino acid sequence of SEQ ID NO:85; (f) theV_(H2) domain comprises the amino acid sequence of SEQ ID NO:49, theV_(L2) domain comprises the amino acid sequence of SEQ ID NO:50, theV_(H1) domain comprises the amino acid sequence of SEQ ID NO:84, and theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:85; (g) theV_(H1) domain comprises the amino acid sequence of SEQ ID NO:51, theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:52, theV_(H2) domain comprises the amino acid sequence of SEQ ID NO:84, and theV_(L2) domain comprises the amino acid sequence of SEQ ID NO:85; or (h)the V_(H2) domain comprises the amino acid sequence of SEQ ID NO:51, theV_(L2) domain comprises the amino acid sequence of SEQ ID NO:52, theV_(H1) domain comprises the amino acid sequence of SEQ ID NO:84, and theV_(L1) domain comprises the amino acid sequence of SEQ ID NO:85.
 56. Thebinding protein of claim 55, wherein (a) the V_(H1) domain comprises theamino acid sequence of SEQ ID NO:49, the V_(L1) domain comprises theamino acid sequence of SEQ ID NO:50, the V_(H2) domain comprises theamino acid sequence of SEQ ID NO:53, the V_(L2) domain comprises theamino acid sequence of SEQ ID NO:54, the V_(H3) domain comprises theamino acid sequence of SEQ ID NO:13, and the V_(L3) domain comprises theamino acid sequence of SEQ ID NO:14; or (b) the V_(H1) domain comprisesthe amino acid sequence of SEQ ID NO:49, the V_(L1) domain comprises theamino acid sequence of SEQ ID NO:50, the V_(H2) domain comprises theamino acid sequence of SEQ ID NO:53, the V_(L2) domain comprises theamino acid sequence of SEQ ID NO:54, the V_(H3) domain comprises theamino acid sequence of SEQ ID NO:9, and the V_(L3) domain comprises theamino acid sequence of SEQ ID NO:10.
 57. The binding protein of any oneof claims 48-56, wherein at least one of L₁, L₂, L₃ or L₄ isindependently 0 amino acids in length.
 58. The binding protein of anyone of claims 48-56, wherein L₁, L₂, L₃ or L₄ are each independently atleast one amino acid in length.
 59. The binding protein of any one ofclaims 48-56, wherein (a) L₁, L₂, L₃ and L₄ each independently are zeroamino acids in length or comprise a sequence selected from the groupconsisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56),S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG(SEQ ID NO:59); or (b) L₁, L₂, L₃ and L₄ each independently comprise asequence selected from the group consisting of GGGGSGGGGS (SEQ IDNO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57),GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59).
 60. The bindingprotein of any one of claims 48-56, wherein (a) L₁ comprises thesequence GQPKAAP (SEQ ID NO: 58), L₂ comprises the sequence TKGPS (SEQID NO:57), L₃ comprises the sequence S, and L₄ comprises the sequenceRT; (b) L₁ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₂comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₃ is 0 amino acids inlength, and L₄ is 0 amino acids in length; (c) L₁ comprises the sequenceGGSGSSGSGG (SEQ ID NO:59), L₂ comprises the sequence GGSGSSGSGG (SEQ IDNO:59), L₃ is 0 amino acids in length, and L₄ is 0 amino acids inlength; or (d) L₁ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56),L₂ is 0 amino acids in length, L₃ comprises the sequence GGGGSGGGGSGGGGS(SEQ ID NO:56), and L₄ is 0 amino acids in length.
 61. The bindingprotein of any one of claims 48-60, wherein the hinge-C_(H2)-C_(H3)domains of the second and the third polypeptide chains are human IgG4hinge-C_(H2)-C_(H3) domains, and wherein the hinge-C_(H2)-C_(H3) domainseach comprise amino acid substitutions at positions corresponding topositions 234 and 235 of human IgG4 according to EU Index, wherein theamino acid substitutions are F234A and L235A.
 62. The binding protein ofany one of claims 48-60, wherein the hinge-C_(H2)-C_(H3) domains of thesecond and the third polypeptide chains are human IgG4hinge-C_(H2)-C_(H3) domains, and wherein the hinge-C_(H2)-C_(H3) domainseach comprise amino acid substitutions at positions corresponding topositions 233-236 of human IgG4 according to EU Index, wherein the aminoacid substitutions are E233P, F234V, L235A, and a deletion at
 236. 63.The binding protein of any one of claims 48-62, wherein thehinge-C_(H2)-C_(H3) domains of the second and the third polypeptidechains are human IgG4 hinge-C_(H2)-C_(H3) domains, and wherein thehinge-C_(H2)-C_(H3) domains each comprise amino acid substitutions atpositions corresponding to positions 228 and 409 of human IgG4 accordingto EU Index, wherein the amino acid substitutions are S228P and R409K.64. The binding protein of any one of claims 48-60, wherein thehinge-C_(H2)-C_(H3) domains of the second and the third polypeptidechains are human IgG1 hinge-C_(H2)-C_(H3) domains, and wherein thehinge-C_(H2)-C_(H3) domains each comprise amino acid substitutions atpositions corresponding to positions 234, 235, and 329 of human IgG1according to EU Index, wherein the amino acid substitutions are L234A,L235A, and P329A.
 65. The binding protein of any one of claims 48-60,wherein the hinge-C_(H2)-C_(H3) domains of the second and the thirdpolypeptide chains are human IgG1 hinge-C_(H2)-C_(H3) domains, andwherein the hinge-C_(H2)-C_(H3) domains each comprise amino acidsubstitutions at positions corresponding to positions 298, 299, and 300of human IgG1 according to EU Index, wherein the amino acidsubstitutions are S298N, T299A, and Y300S.
 66. The binding protein ofany one of claims 48-65, wherein the hinge-C_(H2)-C_(H3) domain of thesecond polypeptide chain comprises amino acid substitutions at positionscorresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4according to EU Index, wherein the amino acid substitutions are Y349C,T366S, L368A, and Y407V; and wherein the hinge-C_(H2)-C_(H3)domain ofthe third polypeptide chain comprises amino acid substitutions atpositions corresponding to positions 354 and 366 of human IgG1 or IgG4according to EU Index, wherein the amino acid substitutions are S354Cand T366W.
 67. The binding protein of any one of claims 48-65, whereinthe hinge-C_(H2)-C_(H3) domain of the second polypeptide chain comprisesamino acid substitutions at positions corresponding to positions 354 and366 of human IgG1 or IgG4 according to EU Index, wherein the amino acidsubstitutions are S354C and T366W; and wherein the hinge-C_(H2)-C_(H3)domain of the third polypeptide chain comprises amino acid substitutionsat positions corresponding to positions 349, 366, 368, and 407 of humanIgG1 or IgG4 according to EU Index, wherein the amino acid substitutionsare Y349C, T366S, L368A, and Y407V.
 68. The binding protein of claim 48or claim 52, wherein the first polypeptide chain comprises the aminoacid sequence of SEQ ID NO:61, the second polypeptide chain comprisesthe amino acid sequence of SEQ ID NO:60, the third polypeptide chaincomprises the amino acid sequence of SEQ ID NO:62, and the fourthpolypeptide chain comprises the amino acid sequence of SEQ ID NO:63. 69.The binding protein of claim 48 or claim 52, wherein the firstpolypeptide chain comprises the amino acid sequence of SEQ ID NO:61, thesecond polypeptide chain comprises the amino acid sequence of SEQ IDNO:64, the third polypeptide chain comprises the amino acid sequence ofSEQ ID NO:65, and the fourth polypeptide chain comprises the amino acidsequence of SEQ ID NO:63.
 70. The binding protein of claim 48 or claim52, wherein the first polypeptide chain comprises the amino acidsequence of SEQ ID NO:61, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:66, the third polypeptide chaincomprises the amino acid sequence of SEQ ID NO:67, and the fourthpolypeptide chain comprises the amino acid sequence of SEQ ID NO:63. 71.The binding protein of claim 48 or claim 52, wherein the firstpolypeptide chain comprises the amino acid sequence of SEQ ID NO:61, thesecond polypeptide chain comprises the amino acid sequence of SEQ IDNO:60, the third polypeptide chain comprises the amino acid sequence ofSEQ ID NO:68, and the fourth polypeptide chain comprises the amino acidsequence of SEQ ID NO:69.
 72. The binding protein of claim 48 or claim52, wherein the first polypeptide chain comprises the amino acidsequence of SEQ ID NO:61, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:64, the third polypeptide chaincomprises the amino acid sequence of SEQ ID NO:70, and the fourthpolypeptide chain comprises the amino acid sequence of SEQ ID NO:69. 73.The binding protein of claim 48 or claim 52, wherein the firstpolypeptide chain comprises the amino acid sequence of SEQ ID NO:61, thesecond polypeptide chain comprises the amino acid sequence of SEQ IDNO:66, the third polypeptide chain comprises the amino acid sequence ofSEQ ID NO:71, and the fourth polypeptide chain comprises the amino acidsequence of SEQ ID NO:69.
 74. A binding protein comprising three antigenbinding sites that each bind one or more target proteins, wherein thebinding protein comprises four polypeptide chains that form the threeantigen binding sites, wherein a first polypeptide chain comprises astructure represented by the formula:V_(L2)-L₁-V_(L1)-L₂-C_(L)  [I] and a second polypeptide chain comprisesa structure represented by the formula:V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3)  [II] and a thirdpolypeptide chain comprises a structure represented by the formula:V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3)  [III] and a fourth polypeptide chaincomprises a structure represented by the formula:V_(L3)-C_(L)  [IV] wherein: V_(L1) is a first immunoglobulin light chainvariable domain; V_(L2) is a second immunoglobulin light chain variabledomain; V_(L3) is a third immunoglobulin light chain variable domain;V_(H1) is a first immunoglobulin heavy chain variable domain; V_(H2) isa second immunoglobulin heavy chain variable domain; V_(H3) is a thirdimmunoglobulin heavy chain variable domain; C_(L) is an immunoglobulinlight chain constant domain; C_(H1) is an immunoglobulin C_(H1) heavychain constant domain; C_(H2) is an immunoglobulin C_(H2) heavy chainconstant domain; C_(H3) is an immunoglobulin C_(H3) heavy chain constantdomain; hinge is an immunoglobulin hinge region connecting the C_(H1)and C_(H2) domains; and L₁, L₂, L₃ and L₄ are amino acid linkers;wherein the polypeptide of formula I and the polypeptide of formula IIform a cross-over light chain-heavy chain pair; and wherein: (a) thehinge-C_(H2)-C_(H3) domains of the second and the third polypeptidechains are human IgG1 hinge-C_(H2)-C_(H3) domains, and wherein thehinge-C_(H2)-C_(H3) domains each comprise amino acid substitutions atpositions corresponding to positions 298, 299, and 300 of human IgG1according to EU Index, wherein the amino acid substitutions are S298N,T299A, and Y300S; or (b) the hinge-C_(H2)-C_(H3) domains of the secondand the third polypeptide chains are human IgG4 C_(H3) domains, andwherein the hinge-C_(H2)-C_(H3) domains each comprise amino acidsubstitutions at positions corresponding to positions 233-236 of humanIgG4 according to EU Index, wherein the amino acid substitutions areE233P, F234V, L235A, and a deletion at
 236. 75. The binding protein ofclaim 74, wherein at least one pair of V_(H1) and V_(L1), V_(H2) andV_(L2), and V_(H3) and V_(L3) forms an antigen binding site that binds aCD38 polypeptide.
 76. The binding protein of claim 74, wherein one, two,or three pairs of V_(H1) and V_(L1), V_(H2) and V_(L2), and V_(H3) andV_(L3) form an antigen binding site that binds an antigen targetselected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR,BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4,C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19,CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23,CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137,CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278,CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3,CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphatediphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24,GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE,IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R,IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1,IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35,ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1,NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152,SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3,TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP,TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.
 77. The bindingprotein of claim 74, wherein a first pair of V_(H1) and V_(L1), V_(H2)and V_(L2), and V_(H3) and V_(L3) forms an antigen binding site thatbinds a human CD3 polypeptide, a second pair of V_(H1) and V_(L1),V_(H2) and V_(L2), and V_(H3) and V_(L3) forms an antigen binding sitethat binds a human CD28 polypeptide, and a third pair of V_(H1) andV_(L1), V_(H2) and V_(L2), and V_(H3) and V_(L3) forms an antigenbinding site that binds a human antigen target selected from the groupconsisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA,B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3,CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24,CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38,CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160,CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9,CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3,DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1,FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2,HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B,IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R,IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18,IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1,leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L,PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2,Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9,TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA,Vstm3, WUCAM, and XCR1.
 78. A kit of polynucleotides, comprising: (a) afirst polynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:72, a thirdpolynucleotide comprising the sequence of SEQ ID NO:74, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:75; (b) a firstpolynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:76, a thirdpolynucleotide comprising the sequence of SEQ ID NO:77, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:75; (c) a firstpolynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:78, a thirdpolynucleotide comprising the sequence of SEQ ID NO:79, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:75; (d) a firstpolynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:72, a thirdpolynucleotide comprising the sequence of SEQ ID NO:80, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:81; (e) a firstpolynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:76, a thirdpolynucleotide comprising the sequence of SEQ ID NO:82, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:81; or (f) a firstpolynucleotide comprising the sequence of SEQ ID NO:73, a secondpolynucleotide comprising the sequence of SEQ ID NO:78, a thirdpolynucleotide comprising the sequence of SEQ ID NO:83, and a fourthpolynucleotide comprising the sequence of SEQ ID NO:81.
 79. Apolynucleotide encoding the binding protein of any one of claims 1-77.80. A vector comprising the polynucleotide of claim
 79. 81. A host cellcomprising the kit of polynucleotides of claim 78, the polynucleotide ofclaim 79, or the vector of claim
 80. 82. A method of producing a bindingprotein, the method comprising culturing the host cell of claim 81 suchthat the binding protein is produced.
 83. The method of claim 82,further comprising recovering the binding protein from the host cell.84. A pharmaceutical composition comprising the binding protein of anyone of claims 1-77 and a pharmaceutically acceptable carrier.
 85. Amethod of preventing and/or treating cancer in a patient comprisingadministering to the patient a therapeutically effective amount of atleast one binding protein of any one of claims 1-73 or thepharmaceutical composition of claim
 84. 86. The method of claim 85,wherein the binding protein comprises one antigen binding site thatbinds a T-cell surface protein and another antigen binding site thatbinds the extracellular domain of a human CD38 polypeptide.
 87. Themethod of claim 86, wherein the binding protein is a trispecific bindingprotein comprising a first antigen binding site that binds CD3, a secondantigen binding site that binds CD28, and a third antigen binding sitethat binds the extracellular domain of a human CD38 polypeptide.
 88. Themethod of any one of claims 85-87, wherein the at least one bindingprotein is co-administered with a chemotherapeutic agent.
 89. The methodof any one of claims 85-88, wherein the cancer is multiple myeloma. 90.The method of any one of claims 85-88, wherein the cancer is acutemyeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chroniclymphocytic leukemia (CLL), or a B cell lymphoma.
 91. The method of anyone of claims 85-90, wherein the patient is a human.
 92. The method ofany one of claims 85-91, wherein the patient is selected for treatmentbecause cells of the cancer express a human CD38 isoform E polypeptideon their cell surface.